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  • 95
    Illumina Inc amplicons
    Representative examples of BiSeqS <t>amplicons</t> prepared for eight genomic loci. Differences in primer length often create longer products on one strand, allowing for easy discrimination of equimolar amplification of both strands.
    Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 17327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc amplicon sequencing
    Potential error sources in next-generation sequencing workflow. a Illustration of the major steps of a typical next-generation sequencing workflow. Targeted deep sequencing is usually done by <t>amplicon</t> protocol or hybridization-capture protocol. Potential error sources are indicated by numbers. b Percentage of high-quality (Q30) bases by position in NGS read. This shows that the first and the last 5 bp have lower percentages of high-quality bases than do other positions. c Cumulative plot of NGS read quality distribution categorized by low-quality mapping (MAPQ
    Amplicon Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 3375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies amplicons
    Long range PCR analysis of mtDNA from individual myofibres from a sIBM patient. A representative agarose gel image of 10-kb long range PCR analysis of DNA extracted from four individual COX-normal (‘cell 1–4’) and COX-deficient myofibres (‘cell 5–8’) from a sIBM patient (‘P8’) is presented. A positive control of DNA extracted from whole-blood of a healthy individual (‘c’) was used to ensure detection of full-length <t>amplicons</t> whereas a no-template (‘nt’) control sample served as a control for contamination. A 10-kb product was amplified with wild-type mtDNA (‘c’) whereas shorter products were formed with mtDNA molecules harbouring deletions (‘cell1-4’). A single deletion species was found in ‘cell 1’ and ‘cell 2’ (blue arrows) whilst ‘cell 3’ and ‘cell 4’ contained two or three deletions of different sizes respectively (red arrows). The location of the forward (‘F’) and reverse (‘R’) primers used in the assay (F6358 and R001) is shown in a schematic representation of an mtDNA molecule on the right.
    Amplicons, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 3253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Macrogen amplicons
    Long range PCR analysis of mtDNA from individual myofibres from a sIBM patient. A representative agarose gel image of 10-kb long range PCR analysis of DNA extracted from four individual COX-normal (‘cell 1–4’) and COX-deficient myofibres (‘cell 5–8’) from a sIBM patient (‘P8’) is presented. A positive control of DNA extracted from whole-blood of a healthy individual (‘c’) was used to ensure detection of full-length <t>amplicons</t> whereas a no-template (‘nt’) control sample served as a control for contamination. A 10-kb product was amplified with wild-type mtDNA (‘c’) whereas shorter products were formed with mtDNA molecules harbouring deletions (‘cell1-4’). A single deletion species was found in ‘cell 1’ and ‘cell 2’ (blue arrows) whilst ‘cell 3’ and ‘cell 4’ contained two or three deletions of different sizes respectively (red arrows). The location of the forward (‘F’) and reverse (‘R’) primers used in the assay (F6358 and R001) is shown in a schematic representation of an mtDNA molecule on the right.
    Amplicons, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 2133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc pcr amplicons
    Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of <t>PCR</t> <t>amplicons</t> spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.
    Pcr Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad amplicons
    DPMT validation for MOB P3 and MOB P4 relaxases. A) Phylogenetic tree of MOB P3 and MOB P4 relaxase families. B) Alignment of the relaxase motifs used to design the MOB P3 and MOB P4 degenerate primers (P3-f+P3-r, continuous black; and P4-f+P4-r, continuous dark grey). C) <t>Amplicons</t> obtained with primers for subfamily MOB P31 (P3-f and P3-r). D) Amplicons obtained with primers for subfamily MOB P42 (P4-f and P4-r). Symbols, colour codes and lanes as in Figure 1 .
    Amplicons, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 3401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher amplicons
    Negative images of electrophoresis gels showing the (A) Clock1a / sdY and (B) D-loop/ sdY PCR assay results for modern male and female samples from five Pacific salmonid species. The approximate location of the IPC and sdY <t>amplicons</t> are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA).
    Amplicons, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 51844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc 16s rrna gene amplicons
    Maximum-likehood (ML) tree based on <t>16S</t> <t>rRNA</t> gene sequences showing the phylogenetic position of Deltaproteobacteria enriched from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values > 75% are indicated at nodes. The bars represent the relative abundance of each OTU affiliated with Deltaproteobacteria in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.
    16s Rrna Gene Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Eurofins amplicons
    HPV DNA detection by PCR amplification, capillary electrophoresis and dideoxy (Sanger) sequencing. a Gel image and electropherogram of amplicon detection by high-resolution capillary electrophoresis. Representative samples #311, 312, 319, and 330 (HSIL) reveal 1 or 2 <t>amplicons</t> after using consensus primers (GP-E6/E7 F/B) to amplify an E6/E7 segment with an expected fragment size of ~660 bp (range, 619-819 bp). Amplicon size variability reflects sequence differences between HPV genotypes. In general, deep sequencing resolved a greater number of HPV genotypes than capillary electrophoresis per sample. Sample #330 illustrates this with detection of 2 amplicons on electrophoresis, but 8 genotypes by deep sequencing. b Representative sample (#311) with a single HPV infection revealing 1 amplicon (699 bp peak on electropherogram) and clean sequencing chromatogram. Representative sample (#319) with multiple HPV infections revealing 2 amplicons (619 and 656 bp peaks on electropherogram) and “noisy” overlapping peaks on the chromatogram. AM, alignment marker; B, buffer; bp, base pair; M, molecular-weight marker
    Amplicons, supplied by Eurofins, used in various techniques. Bioz Stars score: 94/100, based on 1135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    MACHEREY NAGEL amplicons
    Repeat sizes recovered after molecular cloning of <t>amplicons</t> containing uracil or thymidine using DH5α and DH5α Δung strains.
    Amplicons, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 1498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad pcr amplicons
    Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of <t>PCR</t> <t>amplicons</t> spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.
    Pcr Amplicons, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc truseq amplicon cancer panel
    Mutation frequency by gene from results of ( a ) MALDI-TOF, n = 827, and ( b ) <t>TruSeq</t> <t>Amplicon</t> Cancer Panel, n = 792. Mutation frequency was calculated as number of variant occurrences within each gene divided by the total number of patients
    Truseq Amplicon Cancer Panel, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Pacific Biosciences amplicons
    Sequence accuracy as a function of subread depth. a Accuracy of consensus and b assembly sequences. Data from all the <t>amplicons</t> were pooled together to evaluate the consensus calling accuracy as a function of depth of coverage of SMRT raw reads. The vertical line shows the minimum read depth of the consensus sequences used for assemblies
    Amplicons, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Genewiz amplicons
    Efficient correction of OTC-deficient patient-derived primary human hepatocytes in vivo . (A) Experimental overview. Cells were engrafted into FRG mice and repopulation established by cycling on and off the drug 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione. 20 Eleven weeks later, when the mice were on day 11 of a 21-day water cycle, animals received 5×10 10 vg/mouse SaCas9-sgRNA4 and 2×10 11 vg/mouse donor vectors, 2.5×10 10 vg/mouse SaCas9-sgRNA4 and 1×10 11 vg/mouse donor vectors or 1.25×10 10 vg/mouse SaCas9-sgRNA4 and 5×10 10 vg/mouse donor vectors packaged in the NP59 capsid via intraperitoneal delivery (n = 3 per treatment group for controls and highest vector dose, and n = 4 for 2 lower vector doses). Livers were analyzed 5 weeks following vector delivery. Next-generation Illumina® sequencing was performed across the OTC locus from (B, C) genomic DNA and (D, E) cDNA isolated from FRG mice transplanted with patient-derived human hepatocytes to quantitate the HDR rates and characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. Control samples represent PCR <t>amplicons</t> from engrafted FRG mice that did not receive AAV vector treatment. AAV, adeno-associated virus; FRG, Fah -/- Rag2 -/- Il2rg -/ ; HDR, homology-directed repair; InDels, insertions and deletions; ITR, inverted terminal repeat; SaCas9, Staphylococcus aureus Cas9 nuclease; sgRNA, single guide RNA; rAAV, recombinant AAV.
    Amplicons, supplied by Genewiz, used in various techniques. Bioz Stars score: 94/100, based on 710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene amplicons
    (A) SYBR green dissociation curve analyses of LSG-qPCR <t>amplicons</t> from the Stratagene Mx3000P platform. G1, L . ( V .) braziliensis , L . ( V .) guyanensis , and L . ( V .) panamensis ; G2A/B, L . ( L .) infantum/L . ( L .) chagasi and L . ( L .) tropica ; G3, L . ( L .) amazonensis
    Amplicons, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 823 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc 16s rrna amplicon sequencing
    Microbiota diversity is not regained upon direct weaning the diet-switching group onto the high-MAC diet a , Alpha-diversity as measured by Shannon index of fecal microbiota from generation 5 mice from the high-MAC diet control (control) (n=6), generation 5, diet-switching group that was weaned directly onto the high-MAC diet (Gen 5 diet switching) (n=6), and generation 4 mice from the diet switching group after weaning and maintenance on the low-MAC diet for 13 weeks and returned to the high-MAC diet for four weeks (Gen 4 diet switching) (n=5). Error bars are shown as s.e.m. and P values are from a two-tailed Student’s t-test b , Principal coordinate analysis of unweighted UniFrac distance for <t>16S</t> <t>rRNA</t> <t>amplicon</t> profiles from fecal samples collected from first generation control mice on a high-MAC diet (green), fourth generation, diet-switching mice (purple), and fifth generation mice from the diet-switching lineage weaned directly onto the high-MAC diet (orange). Control is plotted as weeks post-humanization and generation 4 and 5 are plotted as age.
    16s Rrna Amplicon Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biotium amplicons
    Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 <t>amplicons</t> served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively
    Amplicons, supplied by Biotium, used in various techniques. Bioz Stars score: 92/100, based on 1146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Illumina Inc miseq amplicon sequencing
    Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 <t>amplicons</t> served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively
    Miseq Amplicon Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Representative examples of BiSeqS amplicons prepared for eight genomic loci. Differences in primer length often create longer products on one strand, allowing for easy discrimination of equimolar amplification of both strands.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: Representative examples of BiSeqS amplicons prepared for eight genomic loci. Differences in primer length often create longer products on one strand, allowing for easy discrimination of equimolar amplification of both strands.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques: Amplification

    BiSeqS drastically reduces the MAF of single base substitution mutations across amplified loci. ( A ) MAF of mutations per position across all amplicons. ( B ) MAF of supermutants per position across all amplicons. ( C ) MAF of SDMs per position across all amplicons.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: BiSeqS drastically reduces the MAF of single base substitution mutations across amplified loci. ( A ) MAF of mutations per position across all amplicons. ( B ) MAF of supermutants per position across all amplicons. ( C ) MAF of SDMs per position across all amplicons.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques: Amplification

    Sensitivity of BiSeqS across all additional amplicons at nominal mutant allele fractions (MAF) of 0.20% and 0.02%. BiSeqS maintains the sensitivity inherent to PCR-based molecular barcoding by detecting mutations at a similar frequency to NGS and molecular barcode-based sequencing.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: Sensitivity of BiSeqS across all additional amplicons at nominal mutant allele fractions (MAF) of 0.20% and 0.02%. BiSeqS maintains the sensitivity inherent to PCR-based molecular barcoding by detecting mutations at a similar frequency to NGS and molecular barcode-based sequencing.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing

    BiSeqS drastically reduces the MAF of indel mutations across amplified loci. ( A ) MAF of mutations per position across all amplicons. ( B ) MAF of supermutants per position across all amplicons. ( C ) MAF of SDMs per position across all amplicons.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: BiSeqS drastically reduces the MAF of indel mutations across amplified loci. ( A ) MAF of mutations per position across all amplicons. ( B ) MAF of supermutants per position across all amplicons. ( C ) MAF of SDMs per position across all amplicons.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques: Amplification

    BiSeqS drastically reduces the number of single base substitution mutations. ( A ) Number of mutations per position across all amplicons. ( B ) Number of supermutants per position across all amplicons. ( C ) Number of SDMs per position across all amplicons. Note that the y axis scales in A and C differ by three orders of magnitude.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: BiSeqS drastically reduces the number of single base substitution mutations. ( A ) Number of mutations per position across all amplicons. ( B ) Number of supermutants per position across all amplicons. ( C ) Number of SDMs per position across all amplicons. Note that the y axis scales in A and C differ by three orders of magnitude.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques:

    BiSeqS drastically reduces the number of indel mutations across amplified loci. ( A ) Number of mutations per position across all amplicons. ( B ) Number of supermutants per position across all amplicons. ( C ) Number of SDMs per position across all amplicons.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

    doi: 10.1073/pnas.1701382114

    Figure Lengend Snippet: BiSeqS drastically reduces the number of indel mutations across amplified loci. ( A ) Number of mutations per position across all amplicons. ( B ) Number of supermutants per position across all amplicons. ( C ) Number of SDMs per position across all amplicons.

    Article Snippet: Sequencing of all of the amplicons described above was performed using an Illumina MiSeq instrument.

    Techniques: Amplification

    Sequence variants inferred by DADA2 compared to the OTUs constructed by UPARSE The merged sequences output by DADA2 are plotted for three Illumina amplicon datasets: (a) Balanced, (b) HMP, and (c) Extreme. Frequency is plotted on the y-axis; Hamming distance to the closest more-abundant sequence on the x-axis. Shapes represent accuracy (Methods). When variants are well separated from other members of the community the sequence variants inferred by DADA2 largely coincide with the OTUs output by UPARSE (black). However, DADA2 resolves additional variation (blue), especially within the UPARSE's OTU radius (dashed line), while outputting fewer spurious sequences (One Off and Other).

    Journal: Nature methods

    Article Title: DADA2: High resolution sample inference from Illumina amplicon data

    doi: 10.1038/nmeth.3869

    Figure Lengend Snippet: Sequence variants inferred by DADA2 compared to the OTUs constructed by UPARSE The merged sequences output by DADA2 are plotted for three Illumina amplicon datasets: (a) Balanced, (b) HMP, and (c) Extreme. Frequency is plotted on the y-axis; Hamming distance to the closest more-abundant sequence on the x-axis. Shapes represent accuracy (Methods). When variants are well separated from other members of the community the sequence variants inferred by DADA2 largely coincide with the OTUs output by UPARSE (black). However, DADA2 resolves additional variation (blue), especially within the UPARSE's OTU radius (dashed line), while outputting fewer spurious sequences (One Off and Other).

    Article Snippet: We compared DADA2 to four algorithms (Methods): UPARSE, an OTU construction algorithm with the best published false-positive results ; MED, an algorithm with the best published fine-scale resolution in Illumina amplicon data ; and the popular mothur (average linkage) and QIIME (uclust) OTU methods , .

    Techniques: Sequencing, Construct, Amplification

    Potential error sources in next-generation sequencing workflow. a Illustration of the major steps of a typical next-generation sequencing workflow. Targeted deep sequencing is usually done by amplicon protocol or hybridization-capture protocol. Potential error sources are indicated by numbers. b Percentage of high-quality (Q30) bases by position in NGS read. This shows that the first and the last 5 bp have lower percentages of high-quality bases than do other positions. c Cumulative plot of NGS read quality distribution categorized by low-quality mapping (MAPQ

    Journal: Genome Biology

    Article Title: Analysis of error profiles in deep next-generation sequencing data

    doi: 10.1186/s13059-019-1659-6

    Figure Lengend Snippet: Potential error sources in next-generation sequencing workflow. a Illustration of the major steps of a typical next-generation sequencing workflow. Targeted deep sequencing is usually done by amplicon protocol or hybridization-capture protocol. Potential error sources are indicated by numbers. b Percentage of high-quality (Q30) bases by position in NGS read. This shows that the first and the last 5 bp have lower percentages of high-quality bases than do other positions. c Cumulative plot of NGS read quality distribution categorized by low-quality mapping (MAPQ

    Article Snippet: We targeted known somatic substitution mutations [ , ] by amplicon sequencing (size of 130 ~ 170 bp) on an Illumina HiSeq 2500 sequencer (abbreviated as HiSeq).

    Techniques: Next-Generation Sequencing, Sequencing, Amplification, Hybridization

    Comparison of sequencing errors with known somatic mutations in deep sequencing data generated from diluted COLO829 cancer cell line. a Error rate ( y -axis) in BRAF V600 amplicon ( x -axis: chr7 positions) under standard pileup (top) and CleanDeepSeq (bottom). A > T errors are shown in red and other errors shown in gray. Known somatic mutation BRAF V600E is shown in purple. Also shown are error rates summarized at sample level by pileup (left panels, “ Methods ”) or CleanDeepSeq (right panels) for 1:1000 dilution ( b ) and 1:5000 dilution ( c ). The 12 possible substitution patterns (first parenthesis) are depicted in rows. Median error rates (log10 scale) are indicated on the left, and sample sizes (number of genomic sites) for the histogram are indicated on the right in the second parenthesis. The x -axis displays the error rate in log10 scale. The designed MAF ladders for the known somatic mutations were depicted using red, blue, and black lines labeled on top, and the known somatic mutations were colored according to their expected MAF. Black arrow: BRAF V600E, which has 4 mutant alleles and 2 wildtype alleles in COLO829, so that at 1:1000 dilution and 1:5000 dilution the expected MAF are 0.002 and 0.0004, respectively (“ Methods ”)

    Journal: Genome Biology

    Article Title: Analysis of error profiles in deep next-generation sequencing data

    doi: 10.1186/s13059-019-1659-6

    Figure Lengend Snippet: Comparison of sequencing errors with known somatic mutations in deep sequencing data generated from diluted COLO829 cancer cell line. a Error rate ( y -axis) in BRAF V600 amplicon ( x -axis: chr7 positions) under standard pileup (top) and CleanDeepSeq (bottom). A > T errors are shown in red and other errors shown in gray. Known somatic mutation BRAF V600E is shown in purple. Also shown are error rates summarized at sample level by pileup (left panels, “ Methods ”) or CleanDeepSeq (right panels) for 1:1000 dilution ( b ) and 1:5000 dilution ( c ). The 12 possible substitution patterns (first parenthesis) are depicted in rows. Median error rates (log10 scale) are indicated on the left, and sample sizes (number of genomic sites) for the histogram are indicated on the right in the second parenthesis. The x -axis displays the error rate in log10 scale. The designed MAF ladders for the known somatic mutations were depicted using red, blue, and black lines labeled on top, and the known somatic mutations were colored according to their expected MAF. Black arrow: BRAF V600E, which has 4 mutant alleles and 2 wildtype alleles in COLO829, so that at 1:1000 dilution and 1:5000 dilution the expected MAF are 0.002 and 0.0004, respectively (“ Methods ”)

    Article Snippet: We targeted known somatic substitution mutations [ , ] by amplicon sequencing (size of 130 ~ 170 bp) on an Illumina HiSeq 2500 sequencer (abbreviated as HiSeq).

    Techniques: Sequencing, Generated, Amplification, Mutagenesis, Labeling

    Error profile in NovaSeq + Q5 dataset generated by StJude ( a , b , c ) and HAIB ( d , e , f ). a , d Error rate ( y -axis) in BRAF V600E amplicon ( x -axis: chr7 positions) under direct pileup (top) and CleanDeepSeq (bottom). Also shown are error rates of the 12 change types across two dilutions: b , e 1:1000 dilution; c , f 1:5000 dilution, see Fig. 2 for legends

    Journal: Genome Biology

    Article Title: Analysis of error profiles in deep next-generation sequencing data

    doi: 10.1186/s13059-019-1659-6

    Figure Lengend Snippet: Error profile in NovaSeq + Q5 dataset generated by StJude ( a , b , c ) and HAIB ( d , e , f ). a , d Error rate ( y -axis) in BRAF V600E amplicon ( x -axis: chr7 positions) under direct pileup (top) and CleanDeepSeq (bottom). Also shown are error rates of the 12 change types across two dilutions: b , e 1:1000 dilution; c , f 1:5000 dilution, see Fig. 2 for legends

    Article Snippet: We targeted known somatic substitution mutations [ , ] by amplicon sequencing (size of 130 ~ 170 bp) on an Illumina HiSeq 2500 sequencer (abbreviated as HiSeq).

    Techniques: Generated, Amplification

    5-Azacytidine and 5-aza-2′-deoxycytidine induce similar patterns of mutation during HIV-1 replication. Using the Illumina sequencing data, G-to-C and C-to-G transversion frequencies were determined at every individual guanine (124 in total) or cytosine (116 in total) position within the sequences of the five amplicons. Mutation frequencies for each amplicon were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases) and are represented as mutations per base pair (m/bp). 5-Aza-C- and 5-aza-dC-induced mutation frequencies were then plotted against each other for each sequence position, and the resulting data were subjected to linear regression and correlation analyses. Data represent averages for three independent biological replicates. R 2 denotes the extent to which the best-fit regression line explains the observed variability in the data; P indicates the significance of the correlation; and m indicates the slope of the best-fit regression line.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

    doi: 10.1128/AAC.03084-15

    Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar patterns of mutation during HIV-1 replication. Using the Illumina sequencing data, G-to-C and C-to-G transversion frequencies were determined at every individual guanine (124 in total) or cytosine (116 in total) position within the sequences of the five amplicons. Mutation frequencies for each amplicon were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases) and are represented as mutations per base pair (m/bp). 5-Aza-C- and 5-aza-dC-induced mutation frequencies were then plotted against each other for each sequence position, and the resulting data were subjected to linear regression and correlation analyses. Data represent averages for three independent biological replicates. R 2 denotes the extent to which the best-fit regression line explains the observed variability in the data; P indicates the significance of the correlation; and m indicates the slope of the best-fit regression line.

    Article Snippet: Samples were prepared for Illumina amplicon sequencing as described previously , but with several minor modifications.

    Techniques: Mutagenesis, Sequencing, Amplification

    5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC 75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant ( P > 0.05).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

    doi: 10.1128/AAC.03084-15

    Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC 75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant ( P > 0.05).

    Article Snippet: Samples were prepared for Illumina amplicon sequencing as described previously , but with several minor modifications.

    Techniques: Mutagenesis, Infection, Purification, Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Amplification

    Heat map and frequency distribution of amplicons in chromosome 12. Twenty samples from 12 lesions from five patients with well-differentiated liposarcomas, representing amplicon-driven sarcomas, were analyzed. a The upper panel, based on the log ratios, shows that the extension of gains (green) and copy-neutral loss of heterozygosity (LOH) is highly similar among different samples from the same patient. Note that samples 10A1–A3 (three samples from primary tumor) and 10C (local recurrence 2; LR2) are more similar to each other than 10A1–3 to 10B or 10B to 10C; the same is true for samples 11B (LR1) and 11D (LR3) in comparison to 11C (LR2). b The lower panel, based on the copy number segmentation, shows the frequency of distinct amplicons in chromosome 12 among the 20 samples. Only two segments in 12q14–15, with a combined length of 856 kb, were amplified in all samples

    Journal: Nature Communications

    Article Title: Different patterns of clonal evolution among different sarcoma subtypes followed for up to 25 years

    doi: 10.1038/s41467-018-06098-0

    Figure Lengend Snippet: Heat map and frequency distribution of amplicons in chromosome 12. Twenty samples from 12 lesions from five patients with well-differentiated liposarcomas, representing amplicon-driven sarcomas, were analyzed. a The upper panel, based on the log ratios, shows that the extension of gains (green) and copy-neutral loss of heterozygosity (LOH) is highly similar among different samples from the same patient. Note that samples 10A1–A3 (three samples from primary tumor) and 10C (local recurrence 2; LR2) are more similar to each other than 10A1–3 to 10B or 10B to 10C; the same is true for samples 11B (LR1) and 11D (LR3) in comparison to 11C (LR2). b The lower panel, based on the copy number segmentation, shows the frequency of distinct amplicons in chromosome 12 among the 20 samples. Only two segments in 12q14–15, with a combined length of 856 kb, were amplified in all samples

    Article Snippet: Paired-end 2 × 151 bp reads were generated from the Amplicon libraries using a NextSeq 500 (Illumina).

    Techniques: Amplification

    Circos plots illustrating different modes of clonal evolution in sarcomas with different genetic backgrounds. a A fusion-driven myxoid liposarcoma (MLS), b an amplicon-driven well-differentiated liposarcoma (WDLS), and c a myxofibrosarcoma (MFS) with a complexly rearranged genome. The red/green inner circles represent the location and amplitude of the allelic imbalances; blue is gain, gray is loss and yellow background indicates loss of heterozygosity. The number of red fields can vary between lesions depending on what is considered the expected number of copies for that lesion in relation to the ploidy level (2 n –3 n

    Journal: Nature Communications

    Article Title: Different patterns of clonal evolution among different sarcoma subtypes followed for up to 25 years

    doi: 10.1038/s41467-018-06098-0

    Figure Lengend Snippet: Circos plots illustrating different modes of clonal evolution in sarcomas with different genetic backgrounds. a A fusion-driven myxoid liposarcoma (MLS), b an amplicon-driven well-differentiated liposarcoma (WDLS), and c a myxofibrosarcoma (MFS) with a complexly rearranged genome. The red/green inner circles represent the location and amplitude of the allelic imbalances; blue is gain, gray is loss and yellow background indicates loss of heterozygosity. The number of red fields can vary between lesions depending on what is considered the expected number of copies for that lesion in relation to the ploidy level (2 n –3 n

    Article Snippet: Paired-end 2 × 151 bp reads were generated from the Amplicon libraries using a NextSeq 500 (Illumina).

    Techniques: Amplification

    Schematic illustration of clonal evolution in 20 sarcomas (C1–C20). C1–C9 are gene fusion-driven myxoid liposarcomas (MLS), C10–C14 are amplicon-driven well-differentiated liposarcomas (WDLS), and C15–C20 are sarcomas with complex genotypes (CXS). a Time intervals (in months) between lesions that were analyzed with regard to chromosomal aberrations and nucleotide level mutations. Each sample is indicated by a filled circle; blue samples were analyzed by whole-exome sequencing (WES), SNP arrays (GCS), and chromosome banding analysis (CA), green samples by GCS and CA, and red samples only by CA; larger filled circles represent lesions from which multiple samples were analyzed for assessment of intratumoral heterogeneity. Each line starts with the primary tumor, followed by local recurrences (LR) and/or metastases (M). b Diagram showing the number of non-synonymous exonic variants (ESV) detected at WES, as well as the extent of shared mutations among different samples and lesions from the same patient. c Diagram showing the number of clonal chromosomal breakpoints detected at GCS and, for MLS also including CB, as well as the extent of shared aberrations among different samples and lesions from the same patient. Figures for C8 and C9 are based on CB only

    Journal: Nature Communications

    Article Title: Different patterns of clonal evolution among different sarcoma subtypes followed for up to 25 years

    doi: 10.1038/s41467-018-06098-0

    Figure Lengend Snippet: Schematic illustration of clonal evolution in 20 sarcomas (C1–C20). C1–C9 are gene fusion-driven myxoid liposarcomas (MLS), C10–C14 are amplicon-driven well-differentiated liposarcomas (WDLS), and C15–C20 are sarcomas with complex genotypes (CXS). a Time intervals (in months) between lesions that were analyzed with regard to chromosomal aberrations and nucleotide level mutations. Each sample is indicated by a filled circle; blue samples were analyzed by whole-exome sequencing (WES), SNP arrays (GCS), and chromosome banding analysis (CA), green samples by GCS and CA, and red samples only by CA; larger filled circles represent lesions from which multiple samples were analyzed for assessment of intratumoral heterogeneity. Each line starts with the primary tumor, followed by local recurrences (LR) and/or metastases (M). b Diagram showing the number of non-synonymous exonic variants (ESV) detected at WES, as well as the extent of shared mutations among different samples and lesions from the same patient. c Diagram showing the number of clonal chromosomal breakpoints detected at GCS and, for MLS also including CB, as well as the extent of shared aberrations among different samples and lesions from the same patient. Figures for C8 and C9 are based on CB only

    Article Snippet: Paired-end 2 × 151 bp reads were generated from the Amplicon libraries using a NextSeq 500 (Illumina).

    Techniques: Amplification, Sequencing

    Microbial community relatedness based on 16S rRNA gene amplicon sequencing as quantified by the weighted UniFrac distance metric. Niche type is denoted by color and sample origin by shape. OV water, exhibit water prior to entry into the denitrification system; interstitial water, water from within the system.

    Journal: Applied and Environmental Microbiology

    Article Title: Broad Phylogenetic Diversity Associated with Nitrogen Loss through Sulfur Oxidation in a Large Public Marine Aquarium

    doi: 10.1128/AEM.01250-18

    Figure Lengend Snippet: Microbial community relatedness based on 16S rRNA gene amplicon sequencing as quantified by the weighted UniFrac distance metric. Niche type is denoted by color and sample origin by shape. OV water, exhibit water prior to entry into the denitrification system; interstitial water, water from within the system.

    Article Snippet: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform .

    Techniques: Amplification, Sequencing

    Proportional abundances of microbial taxa based on 16S rRNA gene amplicon sequencing. The figure shows only those bacterial families averaging greater than 0.01 proportional representation. Aragonite samples have been omitted for clarity. *, family containing Sulfurimonas species; **, family containing Thiobacillus species.

    Journal: Applied and Environmental Microbiology

    Article Title: Broad Phylogenetic Diversity Associated with Nitrogen Loss through Sulfur Oxidation in a Large Public Marine Aquarium

    doi: 10.1128/AEM.01250-18

    Figure Lengend Snippet: Proportional abundances of microbial taxa based on 16S rRNA gene amplicon sequencing. The figure shows only those bacterial families averaging greater than 0.01 proportional representation. Aragonite samples have been omitted for clarity. *, family containing Sulfurimonas species; **, family containing Thiobacillus species.

    Article Snippet: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform .

    Techniques: Amplification, Sequencing

    Proportional abundances of the top 10 most abundant Thiobacillus or Sulfurimonas OTUs based on 16S rRNA gene amplicon sequencing. For each sample, the left bar corresponds to the abundance of Thiobacillus or Sulfurimonas reads as a proportion of the total microbial 16S rRNA pool in either water (blue), sulfur (yellow), or aragonite (green). The right bar, either in shades of purple ( Sulfurimonas ) or orange ( Thiobacillus ), shows the proportional representation of the top 10 most abundant OTUs within each genus.

    Journal: Applied and Environmental Microbiology

    Article Title: Broad Phylogenetic Diversity Associated with Nitrogen Loss through Sulfur Oxidation in a Large Public Marine Aquarium

    doi: 10.1128/AEM.01250-18

    Figure Lengend Snippet: Proportional abundances of the top 10 most abundant Thiobacillus or Sulfurimonas OTUs based on 16S rRNA gene amplicon sequencing. For each sample, the left bar corresponds to the abundance of Thiobacillus or Sulfurimonas reads as a proportion of the total microbial 16S rRNA pool in either water (blue), sulfur (yellow), or aragonite (green). The right bar, either in shades of purple ( Sulfurimonas ) or orange ( Thiobacillus ), shows the proportional representation of the top 10 most abundant OTUs within each genus.

    Article Snippet: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform .

    Techniques: Amplification, Sequencing

    Maximum likelihood phylogeny tree showing the relatedness of the top 20 most abundant Sulfurimonas OTUs (red) based on partial 16S rRNA gene sequences and their proportional abundance in the total 16S rRNA amplicon pool. The tree includes reference sequences identified in NCBI nt database as top matches in BLAST queries of each OTU, as well as RDP sequences representing the Sulfurimonas genus. Campylobacter sequences ( n = 7) from RDP were used as an outgroup. The tree was constructed on the basis of an alignment (256 nt) of a total of 73 sequences using the Kimura 2 model iterated for 1,000 bootstraps.

    Journal: Applied and Environmental Microbiology

    Article Title: Broad Phylogenetic Diversity Associated with Nitrogen Loss through Sulfur Oxidation in a Large Public Marine Aquarium

    doi: 10.1128/AEM.01250-18

    Figure Lengend Snippet: Maximum likelihood phylogeny tree showing the relatedness of the top 20 most abundant Sulfurimonas OTUs (red) based on partial 16S rRNA gene sequences and their proportional abundance in the total 16S rRNA amplicon pool. The tree includes reference sequences identified in NCBI nt database as top matches in BLAST queries of each OTU, as well as RDP sequences representing the Sulfurimonas genus. Campylobacter sequences ( n = 7) from RDP were used as an outgroup. The tree was constructed on the basis of an alignment (256 nt) of a total of 73 sequences using the Kimura 2 model iterated for 1,000 bootstraps.

    Article Snippet: Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform .

    Techniques: Amplification, Construct

    Long range PCR analysis of mtDNA from individual myofibres from a sIBM patient. A representative agarose gel image of 10-kb long range PCR analysis of DNA extracted from four individual COX-normal (‘cell 1–4’) and COX-deficient myofibres (‘cell 5–8’) from a sIBM patient (‘P8’) is presented. A positive control of DNA extracted from whole-blood of a healthy individual (‘c’) was used to ensure detection of full-length amplicons whereas a no-template (‘nt’) control sample served as a control for contamination. A 10-kb product was amplified with wild-type mtDNA (‘c’) whereas shorter products were formed with mtDNA molecules harbouring deletions (‘cell1-4’). A single deletion species was found in ‘cell 1’ and ‘cell 2’ (blue arrows) whilst ‘cell 3’ and ‘cell 4’ contained two or three deletions of different sizes respectively (red arrows). The location of the forward (‘F’) and reverse (‘R’) primers used in the assay (F6358 and R001) is shown in a schematic representation of an mtDNA molecule on the right.

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Long range PCR analysis of mtDNA from individual myofibres from a sIBM patient. A representative agarose gel image of 10-kb long range PCR analysis of DNA extracted from four individual COX-normal (‘cell 1–4’) and COX-deficient myofibres (‘cell 5–8’) from a sIBM patient (‘P8’) is presented. A positive control of DNA extracted from whole-blood of a healthy individual (‘c’) was used to ensure detection of full-length amplicons whereas a no-template (‘nt’) control sample served as a control for contamination. A 10-kb product was amplified with wild-type mtDNA (‘c’) whereas shorter products were formed with mtDNA molecules harbouring deletions (‘cell1-4’). A single deletion species was found in ‘cell 1’ and ‘cell 2’ (blue arrows) whilst ‘cell 3’ and ‘cell 4’ contained two or three deletions of different sizes respectively (red arrows). The location of the forward (‘F’) and reverse (‘R’) primers used in the assay (F6358 and R001) is shown in a schematic representation of an mtDNA molecule on the right.

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Amplification

    Map of mtDNA deletions. Size and position within the mitochondrial genome of 20 types of mtDNA deletions identified by 16-kb long range PCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Identifiers ‘A-P3’ correspond to the respective PCR amplicons in Figure 6 .

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Map of mtDNA deletions. Size and position within the mitochondrial genome of 20 types of mtDNA deletions identified by 16-kb long range PCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Identifiers ‘A-P3’ correspond to the respective PCR amplicons in Figure 6 .

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction

    Map of mtDNA deletions. Size and position within the mitochondrial genome of eight types of mtDNA deletions identified by smPCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Numbers ‘1–8’ correspond to the respective PCR amplicons in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Map of mtDNA deletions. Size and position within the mitochondrial genome of eight types of mtDNA deletions identified by smPCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Numbers ‘1–8’ correspond to the respective PCR amplicons in Figure 2 .

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction

    Analysis of individual molecules of mitochondrial genome using single molecule PCR amplification. Representative smPCR gel images from single cell lysates obtained from three sIBM patients (‘P13’, ‘P9’ and ‘P7’), where each amplified product came from a single molecule of mtDNA. In order to ensure that each amplicon was indeed created from one template, the fraction of positives had to be equal or lower than 0.25 (no more than 1 out of 4 PCR reactions contained a product). Primers hybridizing with the D-Loop region were designed to amplify ∼16 kb of mtDNA and are marked as forward (‘F’) and reverse (‘R’) in a schematic of mtDNA molecule on right. One fragment was generated from samples: ‘P13/6’, ‘P13/7’, ‘P9/9’ and ‘P7/1’ signifying one type of mtDNA deletion (additional smaller amplicons, equidistant from the main amplicon in each lane, are a consequence of mispriming). Samples ‘P9/10’ and ‘P9/11’ both gave rise to three amplicons each indicating three different deletion species per cell (blue horizontal lines indicate different sizes of individual fragments). All 20 amplicons marked with numbers (‘1–8’) were sequenced using Sanger sequencing, whereas two products ‘7’ and two products ‘8’ were also verified using next generation deep sequencing. Amplicons ‘*’ and ‘#’ were detected by agarose gel electrophoresis but not sequenced.

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Analysis of individual molecules of mitochondrial genome using single molecule PCR amplification. Representative smPCR gel images from single cell lysates obtained from three sIBM patients (‘P13’, ‘P9’ and ‘P7’), where each amplified product came from a single molecule of mtDNA. In order to ensure that each amplicon was indeed created from one template, the fraction of positives had to be equal or lower than 0.25 (no more than 1 out of 4 PCR reactions contained a product). Primers hybridizing with the D-Loop region were designed to amplify ∼16 kb of mtDNA and are marked as forward (‘F’) and reverse (‘R’) in a schematic of mtDNA molecule on right. One fragment was generated from samples: ‘P13/6’, ‘P13/7’, ‘P9/9’ and ‘P7/1’ signifying one type of mtDNA deletion (additional smaller amplicons, equidistant from the main amplicon in each lane, are a consequence of mispriming). Samples ‘P9/10’ and ‘P9/11’ both gave rise to three amplicons each indicating three different deletion species per cell (blue horizontal lines indicate different sizes of individual fragments). All 20 amplicons marked with numbers (‘1–8’) were sequenced using Sanger sequencing, whereas two products ‘7’ and two products ‘8’ were also verified using next generation deep sequencing. Amplicons ‘*’ and ‘#’ were detected by agarose gel electrophoresis but not sequenced.

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction, Amplification, Generated, Sequencing, Agarose Gel Electrophoresis

    Validation of P8/1 single myofibre long-range PCR result. A total of 16-kb long range PCR produced two products from ‘P8/1’ DNA extract indicating two types of deletion. This was shown by sequencing of the PCR amplicon. In order to validate it further stepping-in PCR method was applied. ( A ) ‘PCR 3’ used primers flanking the shorter deletion (primers indicated by red arrows). Three outcomes were considered: (i) product of 651 bp as a consequence of amplification of molecule ‘M1’; (ii) absence of product should molecule ‘M2’ harbour the only genuine deletion in the sample; (iii) a wild-type product of 7512 bp. ‘PCR 4’ contained primers flanking the larger deletion (green arrows). The possible outcomes were: (i) a single product of 1645 bp if the only deletion molecule present in the cell was ‘M1’; (ii) two products measuring 1645 and 736 bp if both ‘M1’ and ‘M2’ were present; (iii) wild-type band of 8506 bp. ( B ) Agarose gel showing PCR products from ‘reaction 3’ and ‘reaction 4’ carried out using whole-cell lysates from ‘P8/1’ and healthy control homogenate DNA ‘c5’. The amplicons are labelled with appropriate molecular sizes.

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Validation of P8/1 single myofibre long-range PCR result. A total of 16-kb long range PCR produced two products from ‘P8/1’ DNA extract indicating two types of deletion. This was shown by sequencing of the PCR amplicon. In order to validate it further stepping-in PCR method was applied. ( A ) ‘PCR 3’ used primers flanking the shorter deletion (primers indicated by red arrows). Three outcomes were considered: (i) product of 651 bp as a consequence of amplification of molecule ‘M1’; (ii) absence of product should molecule ‘M2’ harbour the only genuine deletion in the sample; (iii) a wild-type product of 7512 bp. ‘PCR 4’ contained primers flanking the larger deletion (green arrows). The possible outcomes were: (i) a single product of 1645 bp if the only deletion molecule present in the cell was ‘M1’; (ii) two products measuring 1645 and 736 bp if both ‘M1’ and ‘M2’ were present; (iii) wild-type band of 8506 bp. ( B ) Agarose gel showing PCR products from ‘reaction 3’ and ‘reaction 4’ carried out using whole-cell lysates from ‘P8/1’ and healthy control homogenate DNA ‘c5’. The amplicons are labelled with appropriate molecular sizes.

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction, Produced, Sequencing, Amplification, Agarose Gel Electrophoresis

    Detection of mtDNA deletions in homogenate DNA samples by next generation sequencing. ( A ) DNA extracted from ten 20-μm muscle cryosections from two sIBM patients (P1 and P5) and two controls (c6 and c8) was subjected to long range PCR. For the patients, four serial dilutions were used in four reactions to find the concentration that would result in the highest number of amplicons. Selected samples are depicted by red arrows. The schematic on the right shows position of PCR primers used (261F and 16291R). ( B ) Graphical representation of read depth for all sequenced samples. Both controls show almost identical peaks and troughs, whereas the patients differ from controls and one another. Low read depth, clearly visible in certain areas of the mitochondrial genome, indicates mtDNA deletions (depicted by red arrows). ( C ) Graphical representation of all deletions detected in the patients’ mtDNA by analysis of chimeric sequencing fragments. Each horizontal bar shows the deleted portion of the mtDNA genome as represented by the chimeric reads that align to two distinct parts of the mtDNA reference sequence. The x axis shows the nucleotide position on the mitochondrial genome (0–16 569 bp). The y axis depicts cumulative read count, with individual reads ordered from top to bottom by 5' and then 3' breakpoints. All chimeric reads are depicted; those in grey have read counts below 5 and are unlikely to represent deletion species. All other multiple read deletions are colour coded for clarity, with breakpoints annotated on both the main figure and in the legend and the height of each bar representing the read count.

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Detection of mtDNA deletions in homogenate DNA samples by next generation sequencing. ( A ) DNA extracted from ten 20-μm muscle cryosections from two sIBM patients (P1 and P5) and two controls (c6 and c8) was subjected to long range PCR. For the patients, four serial dilutions were used in four reactions to find the concentration that would result in the highest number of amplicons. Selected samples are depicted by red arrows. The schematic on the right shows position of PCR primers used (261F and 16291R). ( B ) Graphical representation of read depth for all sequenced samples. Both controls show almost identical peaks and troughs, whereas the patients differ from controls and one another. Low read depth, clearly visible in certain areas of the mitochondrial genome, indicates mtDNA deletions (depicted by red arrows). ( C ) Graphical representation of all deletions detected in the patients’ mtDNA by analysis of chimeric sequencing fragments. Each horizontal bar shows the deleted portion of the mtDNA genome as represented by the chimeric reads that align to two distinct parts of the mtDNA reference sequence. The x axis shows the nucleotide position on the mitochondrial genome (0–16 569 bp). The y axis depicts cumulative read count, with individual reads ordered from top to bottom by 5' and then 3' breakpoints. All chimeric reads are depicted; those in grey have read counts below 5 and are unlikely to represent deletion species. All other multiple read deletions are colour coded for clarity, with breakpoints annotated on both the main figure and in the legend and the height of each bar representing the read count.

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Next-Generation Sequencing, Polymerase Chain Reaction, Concentration Assay, Sequencing

    PCR-based validation of deletion breakpoints. Sequencing of 16kb PCR products ‘E’ and ‘F’ derived from two separate cells from the same patient (‘P13/1’ and ‘P13/2’) revealed an identical double-deletion in their mitochondrial genome. In order to confirm this phenomenon, stepping-in PCR was designed. ( A ) ‘PCR 1’ used primers flanking the outer breakpoints of the double-deletion, as indicated by red arrows. Three possible scenarios were considered: (i) the double-deletion was real, in which case the expected product would measure 1246 bp; (ii) there was only a single deletion contained between the outer breakpoints, in which case the product would be 617-bp long; (iii) the PCR would only generate a wild-type amplicon of 10 449 bp. ‘PCR 2’ used a forward primer binding to the short fragment of DNA in between the inner breakpoints and the same reverse primer as used in ‘PCR 1’ (primers indicated by green arrows). Again, three scenarios were investigated: (i) the double-deletion was genuine, in which case the product would measure 850 bp; (ii) a single deletion was real, in which case there would be no amplicon as the binding site for the forward primer would have been removed; (iii) only wild-type would amplify, producing a fragment of 6239 bp. ( B ) Agarose gel showing amplified products from ‘PCR 1’ and ‘PCR 2’ performed on whole-cell lysate from ‘P13/1’ and ‘P13/2’. The amplicons are labelled with the molecular sizes expected given identical double-deletion in both molecules. ( C ) Cells ‘P13/1’ and ‘P13/2’ visualized using COX/SDH histochemistry prior to laser-microdissection for downstream mtDNA analyses. ( D ) Five healthy control muscle homogenate DNA samples (‘c3-7’) and homogenate DNA from the same sIBM patient (‘P13’) were subjected to ‘PCRs 1 and 2’. Control samples ‘c3-6’ produced only wild-type amplicons, whereas control ‘c7’ and ‘P13’ produced additional smaller products indicating presence of deleted species of mtDNA.

    Journal: Nucleic Acids Research

    Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: PCR-based validation of deletion breakpoints. Sequencing of 16kb PCR products ‘E’ and ‘F’ derived from two separate cells from the same patient (‘P13/1’ and ‘P13/2’) revealed an identical double-deletion in their mitochondrial genome. In order to confirm this phenomenon, stepping-in PCR was designed. ( A ) ‘PCR 1’ used primers flanking the outer breakpoints of the double-deletion, as indicated by red arrows. Three possible scenarios were considered: (i) the double-deletion was real, in which case the expected product would measure 1246 bp; (ii) there was only a single deletion contained between the outer breakpoints, in which case the product would be 617-bp long; (iii) the PCR would only generate a wild-type amplicon of 10 449 bp. ‘PCR 2’ used a forward primer binding to the short fragment of DNA in between the inner breakpoints and the same reverse primer as used in ‘PCR 1’ (primers indicated by green arrows). Again, three scenarios were investigated: (i) the double-deletion was genuine, in which case the product would measure 850 bp; (ii) a single deletion was real, in which case there would be no amplicon as the binding site for the forward primer would have been removed; (iii) only wild-type would amplify, producing a fragment of 6239 bp. ( B ) Agarose gel showing amplified products from ‘PCR 1’ and ‘PCR 2’ performed on whole-cell lysate from ‘P13/1’ and ‘P13/2’. The amplicons are labelled with the molecular sizes expected given identical double-deletion in both molecules. ( C ) Cells ‘P13/1’ and ‘P13/2’ visualized using COX/SDH histochemistry prior to laser-microdissection for downstream mtDNA analyses. ( D ) Five healthy control muscle homogenate DNA samples (‘c3-7’) and homogenate DNA from the same sIBM patient (‘P13’) were subjected to ‘PCRs 1 and 2’. Control samples ‘c3-6’ produced only wild-type amplicons, whereas control ‘c7’ and ‘P13’ produced additional smaller products indicating presence of deleted species of mtDNA.

    Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

    Techniques: Polymerase Chain Reaction, Sequencing, Derivative Assay, Amplification, Binding Assay, Agarose Gel Electrophoresis, Laser Capture Microdissection, Produced

    Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.

    Journal: Nature biotechnology

    Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

    doi: 10.1038/nbt.3290

    Figure Lengend Snippet: Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.

    Article Snippet: Barcoded PCR amplicons were pooled equimolarly, purified by a spin-column and sequenced on the Illumina MiSeq DNA sequencer platform.

    Techniques: Modification, Plasmid Preparation, Polymerase Chain Reaction, Cleavage Assay

    Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. ( a ) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. ( b ). ( c , d amplicons ( c ) or gene addition by HR at the three loci IL2RG , HBB and CCR5 with synthetic sgRNAs ( d ). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. ( e ) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. ( f ) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. ( g ) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

    Journal: Nature biotechnology

    Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

    doi: 10.1038/nbt.3290

    Figure Lengend Snippet: Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. ( a ) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. ( b ). ( c , d amplicons ( c ) or gene addition by HR at the three loci IL2RG , HBB and CCR5 with synthetic sgRNAs ( d ). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. ( e ) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. ( f ) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. ( g ) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

    Article Snippet: Barcoded PCR amplicons were pooled equimolarly, purified by a spin-column and sequenced on the Illumina MiSeq DNA sequencer platform.

    Techniques: Synthesized, Modification, Sequencing, Plasmid Preparation, Positive Control, Polymerase Chain Reaction, Electroporation

    DPMT validation for MOB P3 and MOB P4 relaxases. A) Phylogenetic tree of MOB P3 and MOB P4 relaxase families. B) Alignment of the relaxase motifs used to design the MOB P3 and MOB P4 degenerate primers (P3-f+P3-r, continuous black; and P4-f+P4-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB P31 (P3-f and P3-r). D) Amplicons obtained with primers for subfamily MOB P42 (P4-f and P4-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB P3 and MOB P4 relaxases. A) Phylogenetic tree of MOB P3 and MOB P4 relaxase families. B) Alignment of the relaxase motifs used to design the MOB P3 and MOB P4 degenerate primers (P3-f+P3-r, continuous black; and P4-f+P4-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB P31 (P3-f and P3-r). D) Amplicons obtained with primers for subfamily MOB P42 (P4-f and P4-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    DPMT validation for MOB P1 relaxases. A) Phylogenetic tree of MOB P1 relaxases. B) Alignment of the relaxase motifs used to design the MOB P1 degenerate primers (P11-f, continuous black; P12-f, continuous dark grey; P131-f, continuous grey; P14-f, continuous light grey; and P1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB P11 (P11-f and P1-r). D) Amplicons obtained with primers for subfamily MOB P12 (P12-f and P1-r). E) Amplicons obtained with primers for subfamily MOB P13 (P131-f and P1-r). F) Amplicons obtained with primers for subfamily MOB P14 (P14-f and P1-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB P1 relaxases. A) Phylogenetic tree of MOB P1 relaxases. B) Alignment of the relaxase motifs used to design the MOB P1 degenerate primers (P11-f, continuous black; P12-f, continuous dark grey; P131-f, continuous grey; P14-f, continuous light grey; and P1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB P11 (P11-f and P1-r). D) Amplicons obtained with primers for subfamily MOB P12 (P12-f and P1-r). E) Amplicons obtained with primers for subfamily MOB P13 (P131-f and P1-r). F) Amplicons obtained with primers for subfamily MOB P14 (P14-f and P1-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    DPMT validation for MOB C relaxases. A) Phylogenetic tree of MOB C relaxase family. B) Alignment of the relaxase motifs used to design the MOB C degenerate primers (C11-f+C11-r, continuous black; C12-f+C12-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB C11 (C11-f and C11-r). D) Amplicons obtained with primers for subfamily MOB C12 (C12-f and C12-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB C relaxases. A) Phylogenetic tree of MOB C relaxase family. B) Alignment of the relaxase motifs used to design the MOB C degenerate primers (C11-f+C11-r, continuous black; C12-f+C12-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB C11 (C11-f and C11-r). D) Amplicons obtained with primers for subfamily MOB C12 (C12-f and C12-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    DPMT validation for MOB P5 relaxases. A) Phylogenetic tree of MOB P5 relaxase family. B) Alignment of the relaxase motifs used to design the MOB P5 degenerate primers (P51-f, continuous black; P52-f, continuous dark grey; P5-r, dashed black; and P53-f+P53-r, continuous grey) C) Amplicons obtained with primers for subfamily MOB P51 (P51-f and P5-r). D) Amplicons obtained with primers for subfamily MOB P52 (P52-f and P5-r). E) Amplicons obtained with primers for subfamily MOB P53 (P53-f and P53-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB P5 relaxases. A) Phylogenetic tree of MOB P5 relaxase family. B) Alignment of the relaxase motifs used to design the MOB P5 degenerate primers (P51-f, continuous black; P52-f, continuous dark grey; P5-r, dashed black; and P53-f+P53-r, continuous grey) C) Amplicons obtained with primers for subfamily MOB P51 (P51-f and P5-r). D) Amplicons obtained with primers for subfamily MOB P52 (P52-f and P5-r). E) Amplicons obtained with primers for subfamily MOB P53 (P53-f and P53-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    DPMT validation for MOB F relaxases. A) Phylogenetic tree of MOB F relaxases. Triangles at the end of the branches represent a compressed group of very similar relaxases ( > 95%). A solid black arrow points to the prototype plasmid for each subfamily. Arrows point to plasmids that experimentally amplified, in spite of containing at least one mismatch in the 12 nucleotides of the CORE sequence. Relaxases contained in our reference collection ( Table 1 ) are denoted by an asterisk. Plasmids detectable by PBRT amplification [19] , [20] , [21] , [22] , [24] , [25] , [27] , [28] , [29] are underlined. New relaxase sequences uncovered by DPMT are shown in red. B) Alignment of the relaxase motifs used to design the MOB F degenerate primers. Colour code: red on yellow = invariant amino acids; blue on blue = strongly conserved; black on green = similar; green on white = weakly similar; black on white = not conserved. Black arrowheads point to the key residues that define the relaxase motifs. Different rectangles embrace the conserved amino acids used to infer the 3′ degenerate core of each oligonucleotide (F11-f, continuous black; F12-f, continuous dark grey; and F1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB F11 (F11-f and F1-r). Lane 1, pSU1588; 2, pSU4280; 3, pSU10013; 4, pSU10014; 5, pSU10017; 6, pSU10018; 7, pSU10021; 8, pSU316; 9, pSU10022; 10, pSU10010; 11, R751; 12, pSU10028; 13, pSU10029; 14, pSU10056; 15, pSU10055; 16, pSU10001; 17, pSU10012; 18, pSU10011; 19, pSU10009; 20, pSU4601; 21, pSU10006; 22, pSU10007; 23, pSU10064; 24, pSU10059; 25, pSU10008; 26, pSU10039; 27, pSU10040; 28, pSU10041; 29, pSU10004; 30, pSU10003; 31, pSU10043; 32, pSU4830; 33, pSU10002; 34, negative control. Lane M, molecular mass marker, HyperLadder IV (Bioline). D) Amplicons obtained with primers for subfamily MOB F12 (F12-f and F1-r). Lanes as in (C).

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB F relaxases. A) Phylogenetic tree of MOB F relaxases. Triangles at the end of the branches represent a compressed group of very similar relaxases ( > 95%). A solid black arrow points to the prototype plasmid for each subfamily. Arrows point to plasmids that experimentally amplified, in spite of containing at least one mismatch in the 12 nucleotides of the CORE sequence. Relaxases contained in our reference collection ( Table 1 ) are denoted by an asterisk. Plasmids detectable by PBRT amplification [19] , [20] , [21] , [22] , [24] , [25] , [27] , [28] , [29] are underlined. New relaxase sequences uncovered by DPMT are shown in red. B) Alignment of the relaxase motifs used to design the MOB F degenerate primers. Colour code: red on yellow = invariant amino acids; blue on blue = strongly conserved; black on green = similar; green on white = weakly similar; black on white = not conserved. Black arrowheads point to the key residues that define the relaxase motifs. Different rectangles embrace the conserved amino acids used to infer the 3′ degenerate core of each oligonucleotide (F11-f, continuous black; F12-f, continuous dark grey; and F1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB F11 (F11-f and F1-r). Lane 1, pSU1588; 2, pSU4280; 3, pSU10013; 4, pSU10014; 5, pSU10017; 6, pSU10018; 7, pSU10021; 8, pSU316; 9, pSU10022; 10, pSU10010; 11, R751; 12, pSU10028; 13, pSU10029; 14, pSU10056; 15, pSU10055; 16, pSU10001; 17, pSU10012; 18, pSU10011; 19, pSU10009; 20, pSU4601; 21, pSU10006; 22, pSU10007; 23, pSU10064; 24, pSU10059; 25, pSU10008; 26, pSU10039; 27, pSU10040; 28, pSU10041; 29, pSU10004; 30, pSU10003; 31, pSU10043; 32, pSU4830; 33, pSU10002; 34, negative control. Lane M, molecular mass marker, HyperLadder IV (Bioline). D) Amplicons obtained with primers for subfamily MOB F12 (F12-f and F1-r). Lanes as in (C).

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques: Plasmid Preparation, Amplification, Sequencing, Negative Control, Marker

    DPMT validation for MOB Q relaxases. A) Phylogenetic tree of MOB Q relaxase family. B) Alignment of the relaxase motifs used to design the MOB Q degenerate primers (Q11-f+Q11-r, continuous black; Q12-f+Q12-r, continuous dark grey; and Qu-f+Qu-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB Q11 (Q11-f and Q11-r). D) Amplicons obtained with primers for subfamily MOB Q12 (Q12-f and Q12-r). E) Amplicons obtained with primers for subfamily MOB Qu (Qu-f and Qu-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB Q relaxases. A) Phylogenetic tree of MOB Q relaxase family. B) Alignment of the relaxase motifs used to design the MOB Q degenerate primers (Q11-f+Q11-r, continuous black; Q12-f+Q12-r, continuous dark grey; and Qu-f+Qu-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB Q11 (Q11-f and Q11-r). D) Amplicons obtained with primers for subfamily MOB Q12 (Q12-f and Q12-r). E) Amplicons obtained with primers for subfamily MOB Qu (Qu-f and Qu-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    DPMT validation for MOB H relaxases. A) Phylogenetic tree of MOB H relaxase family. B) Alignment of the relaxase motifs used to design the MOB H degenerate primers (H11-f+H11-r, continuous black; H121-f+H121-r, continuous dark grey; and H2-f+H2-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB H11 (H11-f and H11-r). D) Amplicons obtained with primers for subfamily MOB H121 (H121-f and H121-r). E) Amplicons obtained with primers for subfamily MOB H2 (H2-f and H2-r). Symbols, colour codes and lanes as in Figure 1 .

    Journal: PLoS ONE

    Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    doi: 10.1371/journal.pone.0040438

    Figure Lengend Snippet: DPMT validation for MOB H relaxases. A) Phylogenetic tree of MOB H relaxase family. B) Alignment of the relaxase motifs used to design the MOB H degenerate primers (H11-f+H11-r, continuous black; H121-f+H121-r, continuous dark grey; and H2-f+H2-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB H11 (H11-f and H11-r). D) Amplicons obtained with primers for subfamily MOB H121 (H121-f and H121-r). E) Amplicons obtained with primers for subfamily MOB H2 (H2-f and H2-r). Symbols, colour codes and lanes as in Figure 1 .

    Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

    Techniques:

    Negative images of electrophoresis gels showing the (A) Clock1a / sdY and (B) D-loop/ sdY PCR assay results for modern male and female samples from five Pacific salmonid species. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA).

    Journal: PLoS ONE

    Article Title: An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains

    doi: 10.1371/journal.pone.0193212

    Figure Lengend Snippet: Negative images of electrophoresis gels showing the (A) Clock1a / sdY and (B) D-loop/ sdY PCR assay results for modern male and female samples from five Pacific salmonid species. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA).

    Article Snippet: The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). (PDF) Click here for additional data file.

    Techniques: Electrophoresis, Polymerase Chain Reaction

    Negative images of electrophoresis gels showing the (A) Clock1a / sdY (B) D-loop/ sdY assay results for nine of the analyzed archaeological salmonid samples. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). Note: For SB11, the D-loop/ sdY assay (B) produced two weak nonspecific bands only slightly smaller than the predicted size of the sdY amplicon, suggesting they might represent sdY . However, the Clock1a / sdY assay (A) only yielded a fragment of Clock1a , confirming the nonspecific bands likely do not represent sdY , verifying SB11’s female identity.

    Journal: PLoS ONE

    Article Title: An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains

    doi: 10.1371/journal.pone.0193212

    Figure Lengend Snippet: Negative images of electrophoresis gels showing the (A) Clock1a / sdY (B) D-loop/ sdY assay results for nine of the analyzed archaeological salmonid samples. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). Note: For SB11, the D-loop/ sdY assay (B) produced two weak nonspecific bands only slightly smaller than the predicted size of the sdY amplicon, suggesting they might represent sdY . However, the Clock1a / sdY assay (A) only yielded a fragment of Clock1a , confirming the nonspecific bands likely do not represent sdY , verifying SB11’s female identity.

    Article Snippet: The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). (PDF) Click here for additional data file.

    Techniques: Electrophoresis, Produced, Amplification

    Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Deltaproteobacteria enriched from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values > 75% are indicated at nodes. The bars represent the relative abundance of each OTU affiliated with Deltaproteobacteria in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.

    Journal: Frontiers in Microbiology

    Article Title: Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia)

    doi: 10.3389/fmicb.2017.01583

    Figure Lengend Snippet: Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Deltaproteobacteria enriched from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values > 75% are indicated at nodes. The bars represent the relative abundance of each OTU affiliated with Deltaproteobacteria in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.

    Article Snippet: 16S rRNA sequencing analysis The mixtures of 16S rRNA gene amplicons were generated using a 341F/815R primer set, as previously described by Dowd et al. , and were sequenced by the MiSeq Illumina (paired-end 2 × 300 bp) platform of the Molecular Research Laboratory (Texas, USA).

    Techniques:

    Compositions of microbial communities in sulfate-reducing enrichment cultures from marine sediment using different electron donors and sulfate sources after 14 days. Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities. The names of enrichment cultures (duplicates 1 and 2) have been abbreviated as follows: SF, SL, SA, and SC for Na 2 SO 4 enrichment cultures with formate, lactate, acetate and without electron donor, respectively; SPF, SPL, SPA, and SPC for PG enrichment cultures with formate, lactate, acetate, and without electron donor, respectively.

    Journal: Frontiers in Microbiology

    Article Title: Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia)

    doi: 10.3389/fmicb.2017.01583

    Figure Lengend Snippet: Compositions of microbial communities in sulfate-reducing enrichment cultures from marine sediment using different electron donors and sulfate sources after 14 days. Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities. The names of enrichment cultures (duplicates 1 and 2) have been abbreviated as follows: SF, SL, SA, and SC for Na 2 SO 4 enrichment cultures with formate, lactate, acetate and without electron donor, respectively; SPF, SPL, SPA, and SPC for PG enrichment cultures with formate, lactate, acetate, and without electron donor, respectively.

    Article Snippet: 16S rRNA sequencing analysis The mixtures of 16S rRNA gene amplicons were generated using a 341F/815R primer set, as previously described by Dowd et al. , and were sequenced by the MiSeq Illumina (paired-end 2 × 300 bp) platform of the Molecular Research Laboratory (Texas, USA).

    Techniques:

    Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Firmicutes from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values > 75% are indicated at nodes. The bars represent the relative abundance (in %) of each OTU affiliated with Firmicutes in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.

    Journal: Frontiers in Microbiology

    Article Title: Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia)

    doi: 10.3389/fmicb.2017.01583

    Figure Lengend Snippet: Maximum-likehood (ML) tree based on 16S rRNA gene sequences showing the phylogenetic position of Firmicutes from marine sediment (as inoculum) using different electron donors and sulfate sources (sodium sulfate or phosphogypsum). Representative sequences in the tree were obtained from GenBank (accession number in the brackets). Bootstrap values > 75% are indicated at nodes. The bars represent the relative abundance (in %) of each OTU affiliated with Firmicutes in the enrichment cultures. The blue bars indicate the relative abundance of OTUs in sodium sulfate cultures, whereas the red bars represent the relative abundance of OTUs in PG cultures.

    Article Snippet: 16S rRNA sequencing analysis The mixtures of 16S rRNA gene amplicons were generated using a 341F/815R primer set, as previously described by Dowd et al. , and were sequenced by the MiSeq Illumina (paired-end 2 × 300 bp) platform of the Molecular Research Laboratory (Texas, USA).

    Techniques:

    Compositions of microbial communities in the coastal marine sediment and phosphogypsum samples of Sfax (Tunisia). Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities of marine sediment (MS) and phosphogypsum (PG).

    Journal: Frontiers in Microbiology

    Article Title: Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia)

    doi: 10.3389/fmicb.2017.01583

    Figure Lengend Snippet: Compositions of microbial communities in the coastal marine sediment and phosphogypsum samples of Sfax (Tunisia). Relative phylogenetic abundance was based on frequencies of 16S rRNA gene sequences affiliated with Archaea and major bacterial phyla or proteobacterial classes in the microbial communities of marine sediment (MS) and phosphogypsum (PG).

    Article Snippet: 16S rRNA sequencing analysis The mixtures of 16S rRNA gene amplicons were generated using a 341F/815R primer set, as previously described by Dowd et al. , and were sequenced by the MiSeq Illumina (paired-end 2 × 300 bp) platform of the Molecular Research Laboratory (Texas, USA).

    Techniques: Mass Spectrometry

    Piphillin results comparing 16S rRNA sequence analysis approaches using the BioCyc database. a 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes. Percentage of amplicon sequences from two datasets using two different 16S rRNA sequence analysis approaches passing identity cutoffs from 75 to 100% against 16S rRNA gene sequences in the BioCyc genome database. b Spearman’s correlation coefficient between Piphillin results and shotgun metagenomics at ten different identity cutoffs tested in Piphillin. Spearman’s correlation coefficient was calculated for each sample and mean, 1st and 3rd quartiles are depicted by the boxes. Whiskers extend to the furthest points within 150% of the interquartile range. c Balanced accuracy in identifying differentially abundant features from Piphillin against corresponding metagenomics at each identity cutoff. * indicates p

    Journal: BMC Genomics

    Article Title: Piphillin predicts metagenomic composition and dynamics from DADA2-corrected 16S rDNA sequences

    doi: 10.1186/s12864-019-6427-1

    Figure Lengend Snippet: Piphillin results comparing 16S rRNA sequence analysis approaches using the BioCyc database. a 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes. Percentage of amplicon sequences from two datasets using two different 16S rRNA sequence analysis approaches passing identity cutoffs from 75 to 100% against 16S rRNA gene sequences in the BioCyc genome database. b Spearman’s correlation coefficient between Piphillin results and shotgun metagenomics at ten different identity cutoffs tested in Piphillin. Spearman’s correlation coefficient was calculated for each sample and mean, 1st and 3rd quartiles are depicted by the boxes. Whiskers extend to the furthest points within 150% of the interquartile range. c Balanced accuracy in identifying differentially abundant features from Piphillin against corresponding metagenomics at each identity cutoff. * indicates p

    Article Snippet: Human feces dataset: 16S rRNA PCR and sequencing Library preparation for 16S rRNA gene amplicon sequencing was performed following the Illumina (San Diego, CA, USA) recommendations with some modifications.

    Techniques: Sequencing, Amplification

    Updated references results in more sequences considered in Piphillin analysis. Comparison of 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes based on database version for Piphillin results from ( a ) DADA2-corrected ASVs and ( b ) 97% de novo OTUs

    Journal: BMC Genomics

    Article Title: Piphillin predicts metagenomic composition and dynamics from DADA2-corrected 16S rDNA sequences

    doi: 10.1186/s12864-019-6427-1

    Figure Lengend Snippet: Updated references results in more sequences considered in Piphillin analysis. Comparison of 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes based on database version for Piphillin results from ( a ) DADA2-corrected ASVs and ( b ) 97% de novo OTUs

    Article Snippet: Human feces dataset: 16S rRNA PCR and sequencing Library preparation for 16S rRNA gene amplicon sequencing was performed following the Illumina (San Diego, CA, USA) recommendations with some modifications.

    Techniques: Amplification

    Piphillin results comparing 16S rRNA sequence analysis approaches using the KEGG database. a 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes. Percentage of amplicon sequences from two datasets using two different 16S rRNA sequence analysis approaches passing identity cutoffs from 75 to 100% against 16S rRNA gene sequences in the KEGG genome database. b Spearman’s correlation coefficient between Piphillin results and shotgun metagenomics at ten different identity cutoffs tested in Piphillin. Spearman’s correlation coefficient was calculated for each sample and mean, 1st and 3rd quartiles are depicted by the boxes. Whiskers extend to the furthest points within 150% of the interquartile range. c Balanced accuracy in identifying differentially abundant KOs from Piphillin against corresponding metagenomics at each identity cutoff. * indicates p

    Journal: BMC Genomics

    Article Title: Piphillin predicts metagenomic composition and dynamics from DADA2-corrected 16S rDNA sequences

    doi: 10.1186/s12864-019-6427-1

    Figure Lengend Snippet: Piphillin results comparing 16S rRNA sequence analysis approaches using the KEGG database. a 16S rRNA gene amplicon sequences passing the identity threshold to the reference genomes. Percentage of amplicon sequences from two datasets using two different 16S rRNA sequence analysis approaches passing identity cutoffs from 75 to 100% against 16S rRNA gene sequences in the KEGG genome database. b Spearman’s correlation coefficient between Piphillin results and shotgun metagenomics at ten different identity cutoffs tested in Piphillin. Spearman’s correlation coefficient was calculated for each sample and mean, 1st and 3rd quartiles are depicted by the boxes. Whiskers extend to the furthest points within 150% of the interquartile range. c Balanced accuracy in identifying differentially abundant KOs from Piphillin against corresponding metagenomics at each identity cutoff. * indicates p

    Article Snippet: Human feces dataset: 16S rRNA PCR and sequencing Library preparation for 16S rRNA gene amplicon sequencing was performed following the Illumina (San Diego, CA, USA) recommendations with some modifications.

    Techniques: Sequencing, Amplification

    HPV DNA detection by PCR amplification, capillary electrophoresis and dideoxy (Sanger) sequencing. a Gel image and electropherogram of amplicon detection by high-resolution capillary electrophoresis. Representative samples #311, 312, 319, and 330 (HSIL) reveal 1 or 2 amplicons after using consensus primers (GP-E6/E7 F/B) to amplify an E6/E7 segment with an expected fragment size of ~660 bp (range, 619-819 bp). Amplicon size variability reflects sequence differences between HPV genotypes. In general, deep sequencing resolved a greater number of HPV genotypes than capillary electrophoresis per sample. Sample #330 illustrates this with detection of 2 amplicons on electrophoresis, but 8 genotypes by deep sequencing. b Representative sample (#311) with a single HPV infection revealing 1 amplicon (699 bp peak on electropherogram) and clean sequencing chromatogram. Representative sample (#319) with multiple HPV infections revealing 2 amplicons (619 and 656 bp peaks on electropherogram) and “noisy” overlapping peaks on the chromatogram. AM, alignment marker; B, buffer; bp, base pair; M, molecular-weight marker

    Journal: BMC Genomics

    Article Title: Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix

    doi: 10.1186/s12864-017-3612-y

    Figure Lengend Snippet: HPV DNA detection by PCR amplification, capillary electrophoresis and dideoxy (Sanger) sequencing. a Gel image and electropherogram of amplicon detection by high-resolution capillary electrophoresis. Representative samples #311, 312, 319, and 330 (HSIL) reveal 1 or 2 amplicons after using consensus primers (GP-E6/E7 F/B) to amplify an E6/E7 segment with an expected fragment size of ~660 bp (range, 619-819 bp). Amplicon size variability reflects sequence differences between HPV genotypes. In general, deep sequencing resolved a greater number of HPV genotypes than capillary electrophoresis per sample. Sample #330 illustrates this with detection of 2 amplicons on electrophoresis, but 8 genotypes by deep sequencing. b Representative sample (#311) with a single HPV infection revealing 1 amplicon (699 bp peak on electropherogram) and clean sequencing chromatogram. Representative sample (#319) with multiple HPV infections revealing 2 amplicons (619 and 656 bp peaks on electropherogram) and “noisy” overlapping peaks on the chromatogram. AM, alignment marker; B, buffer; bp, base pair; M, molecular-weight marker

    Article Snippet: Dideoxy sequencing of the amplicons (~200 ng DNA/sample) was performed using primer GP-E6-3 F at Eurofins Operon (USA).

    Techniques: Polymerase Chain Reaction, Amplification, Electrophoresis, Sequencing, Infection, Marker, Molecular Weight

    HPV genotype composition found in LSIL and HSIL samples. Deep sequencing of HPV E6/E7 amplicons derived from each LSIL or HSIL sample identified 1 to 8 HPV genotypes and quantitated their composition (%) based on number of mapped reads to total mapped reads. The top three dominant (highest proportion) genotypes found in LSIL were HPV-39, -16, and -35 [ red , solid/hashed]. The carcinogenicity of LSIL dominant genotypes were: carcinogenic 29/43 (67%, red ); possibly carcinogenic 6/43 (14%, blue ); and not classifiable/probably not carcinogenic 8/43 (19%, green ). For HSIL, the dominant genotype was primarily HPV-16 (21/29, 72%); and the dominant genotypes were all carcinogenic 29/29 (100%, red ). The HPV carcinogenicity is based on IARC’s classification of human carcinogens [ 8 ]. HSIL, high-grade squamous intraepithelial lesion; IARC, International Agency for Research on Cancer; LSIL, low-grade squamous intraepithelial lesion; ID, identification

    Journal: BMC Genomics

    Article Title: Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix

    doi: 10.1186/s12864-017-3612-y

    Figure Lengend Snippet: HPV genotype composition found in LSIL and HSIL samples. Deep sequencing of HPV E6/E7 amplicons derived from each LSIL or HSIL sample identified 1 to 8 HPV genotypes and quantitated their composition (%) based on number of mapped reads to total mapped reads. The top three dominant (highest proportion) genotypes found in LSIL were HPV-39, -16, and -35 [ red , solid/hashed]. The carcinogenicity of LSIL dominant genotypes were: carcinogenic 29/43 (67%, red ); possibly carcinogenic 6/43 (14%, blue ); and not classifiable/probably not carcinogenic 8/43 (19%, green ). For HSIL, the dominant genotype was primarily HPV-16 (21/29, 72%); and the dominant genotypes were all carcinogenic 29/29 (100%, red ). The HPV carcinogenicity is based on IARC’s classification of human carcinogens [ 8 ]. HSIL, high-grade squamous intraepithelial lesion; IARC, International Agency for Research on Cancer; LSIL, low-grade squamous intraepithelial lesion; ID, identification

    Article Snippet: Dideoxy sequencing of the amplicons (~200 ng DNA/sample) was performed using primer GP-E6-3 F at Eurofins Operon (USA).

    Techniques: Sequencing, Derivative Assay

    Repeat sizes recovered after molecular cloning of amplicons containing uracil or thymidine using DH5α and DH5α Δung strains.

    Journal: Scientific Reports

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

    doi: 10.1038/s41598-017-18168-2

    Figure Lengend Snippet: Repeat sizes recovered after molecular cloning of amplicons containing uracil or thymidine using DH5α and DH5α Δung strains.

    Article Snippet: The amplicons were run on an agarose gel, the band was cut out and purified using the Nucleospin Gel and PCR clean up kit from Macherey-Nagel.

    Techniques: Molecular Cloning

    Using heat-labile Ung and/or Proteinase K prevents the degradation of uracil-containing amplicons. ( A ) Experimental scheme used. ( B ) Agarose gel of amplicons from genomic DNA of GFP(CAG) 101 containing uracil and treated post-PCR with (lanes 7–11) or without (lanes 2–6) proteinase K three days after the PCR was performed (lanes 3–6 and 8–11) or immediately after the PCR (fresh, lanes 2, 7). The samples were stored at −20 °C (lanes 3, 8), 4 °C (lanes 4, 9), room temperature (RT°, lanes 5, 10), or were amplified using a heat labile (hl) Ung instead of the conventional Ung and stored at room temperature (lanes 6,11). MW = molecular weight marker (lanes 1 and 12).

    Journal: Scientific Reports

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

    doi: 10.1038/s41598-017-18168-2

    Figure Lengend Snippet: Using heat-labile Ung and/or Proteinase K prevents the degradation of uracil-containing amplicons. ( A ) Experimental scheme used. ( B ) Agarose gel of amplicons from genomic DNA of GFP(CAG) 101 containing uracil and treated post-PCR with (lanes 7–11) or without (lanes 2–6) proteinase K three days after the PCR was performed (lanes 3–6 and 8–11) or immediately after the PCR (fresh, lanes 2, 7). The samples were stored at −20 °C (lanes 3, 8), 4 °C (lanes 4, 9), room temperature (RT°, lanes 5, 10), or were amplified using a heat labile (hl) Ung instead of the conventional Ung and stored at room temperature (lanes 6,11). MW = molecular weight marker (lanes 1 and 12).

    Article Snippet: The amplicons were run on an agarose gel, the band was cut out and purified using the Nucleospin Gel and PCR clean up kit from Macherey-Nagel.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    Plasmids cloned into ung − cells contain undetectable levels of uracil. ( A ) Experimental scheme used. ( B ) Gel of plasmids isolated from ung + cells transformed with a thymine containing insert (black – lanes 2, 5–8) or ung − cells transformed with dTTP (green – 3, 9–12) or dUTP (blue – 4, 13–16). The samples remained untreated (lanes 2–4) or were incubated with 0.5U of Ung (lanes 5–16). Controls for Ung digestion consist of SUMO1 cDNA amplicons containing uracil digested with Ung (lane 17) or left untreated (lane 18). MW = molecular weight markers (lanes 1, 19). The numbers above the lanes refer to the name of the clones tested.

    Journal: Scientific Reports

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

    doi: 10.1038/s41598-017-18168-2

    Figure Lengend Snippet: Plasmids cloned into ung − cells contain undetectable levels of uracil. ( A ) Experimental scheme used. ( B ) Gel of plasmids isolated from ung + cells transformed with a thymine containing insert (black – lanes 2, 5–8) or ung − cells transformed with dTTP (green – 3, 9–12) or dUTP (blue – 4, 13–16). The samples remained untreated (lanes 2–4) or were incubated with 0.5U of Ung (lanes 5–16). Controls for Ung digestion consist of SUMO1 cDNA amplicons containing uracil digested with Ung (lane 17) or left untreated (lane 18). MW = molecular weight markers (lanes 1, 19). The numbers above the lanes refer to the name of the clones tested.

    Article Snippet: The amplicons were run on an agarose gel, the band was cut out and purified using the Nucleospin Gel and PCR clean up kit from Macherey-Nagel.

    Techniques: Clone Assay, Isolation, Transformation Assay, Incubation, Molecular Weight

    Small-pool PCR of expanded repeats using Ung and dUTP. ( A ) SP-PCR blot from reactions pretreated with the standard Ung, amplified with dUTP, and stopped using Proteinase K. The visible bands are shorter than in the samples seen in B., and the smearing below the bands is evident. Each lane contains the product of a PCR with 50 pg of template DNA derived from GFP(CAG) 101 treated with sgCTG and the Cas9 nickase. ( B ) SP-PCR blots of amplicons generated using dTTP or dUTP, pretreated with hl Ung, and stopped after the PCR with Proteinase K. Each lane contains the product of a PCR that used 250 pg of genomic DNA from GFP(CAG) 101 treated with the Cas9 nickase and either the sgCTG or an empty guide RNA vector (pPN10). In all cases, the probe used was generated with dUTP.

    Journal: Scientific Reports

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

    doi: 10.1038/s41598-017-18168-2

    Figure Lengend Snippet: Small-pool PCR of expanded repeats using Ung and dUTP. ( A ) SP-PCR blot from reactions pretreated with the standard Ung, amplified with dUTP, and stopped using Proteinase K. The visible bands are shorter than in the samples seen in B., and the smearing below the bands is evident. Each lane contains the product of a PCR with 50 pg of template DNA derived from GFP(CAG) 101 treated with sgCTG and the Cas9 nickase. ( B ) SP-PCR blots of amplicons generated using dTTP or dUTP, pretreated with hl Ung, and stopped after the PCR with Proteinase K. Each lane contains the product of a PCR that used 250 pg of genomic DNA from GFP(CAG) 101 treated with the Cas9 nickase and either the sgCTG or an empty guide RNA vector (pPN10). In all cases, the probe used was generated with dUTP.

    Article Snippet: The amplicons were run on an agarose gel, the band was cut out and purified using the Nucleospin Gel and PCR clean up kit from Macherey-Nagel.

    Techniques: Polymerase Chain Reaction, Amplification, Derivative Assay, Generated, Plasmid Preparation

    dTTP substituted with dUTP allows PCR amplification of expanded CAG repeats. ( A ) Experimental scheme used to generate the PCR amplicons found in B. gDNA = genomic DNA ( B ) Agarose gel of PCR amplicons after pre-treatment with Ung and containing either dTTP (lanes 2–5) or dUTP (lanes 6–9) from genomic DNA isolated from GFP(CAG) 15 (lanes 2, 6), GFP(CAG) 50 (lanes 3, 7), GFP(CAG) 101 (lanes 4, 8), or without DNA added (lanes 5, 9). MW = molecular weight marker (lanes 1, 10). ( C ) Experimental scheme used for panel D. ( D ) Agarose gel of PCR amplicons obtained after pre-treatment with Ung of a template containing either dTTP (lanes 2–9) or dUTP (lanes 10–17). The template was generated from amplifying GFP(CAG) 101 genomic DNA. The PCRs with no Ung treatment (lanes 3, 11) contained 1X concentration of the template amplicon. Ten-fold dilution series (lanes 4–8 et 12–16) of the template (20X loaded directly without PCR in (lanes 2, 10)) and no DNA controls are also shown (lanes 9 and 17). MW = molecular weight marker (lanes 1, 18).

    Journal: Scientific Reports

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

    doi: 10.1038/s41598-017-18168-2

    Figure Lengend Snippet: dTTP substituted with dUTP allows PCR amplification of expanded CAG repeats. ( A ) Experimental scheme used to generate the PCR amplicons found in B. gDNA = genomic DNA ( B ) Agarose gel of PCR amplicons after pre-treatment with Ung and containing either dTTP (lanes 2–5) or dUTP (lanes 6–9) from genomic DNA isolated from GFP(CAG) 15 (lanes 2, 6), GFP(CAG) 50 (lanes 3, 7), GFP(CAG) 101 (lanes 4, 8), or without DNA added (lanes 5, 9). MW = molecular weight marker (lanes 1, 10). ( C ) Experimental scheme used for panel D. ( D ) Agarose gel of PCR amplicons obtained after pre-treatment with Ung of a template containing either dTTP (lanes 2–9) or dUTP (lanes 10–17). The template was generated from amplifying GFP(CAG) 101 genomic DNA. The PCRs with no Ung treatment (lanes 3, 11) contained 1X concentration of the template amplicon. Ten-fold dilution series (lanes 4–8 et 12–16) of the template (20X loaded directly without PCR in (lanes 2, 10)) and no DNA controls are also shown (lanes 9 and 17). MW = molecular weight marker (lanes 1, 18).

    Article Snippet: The amplicons were run on an agarose gel, the band was cut out and purified using the Nucleospin Gel and PCR clean up kit from Macherey-Nagel.

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Isolation, Molecular Weight, Marker, Generated, Concentration Assay

    Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.

    Journal: Nature biotechnology

    Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

    doi: 10.1038/nbt.3290

    Figure Lengend Snippet: Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.

    Article Snippet: PCR amplicons spanning the sgRNA genomic target sites were generated using the iProof High-Fidelity Master Mix (Bio-Rad, Hercules, CA, USA) with the following primer pairs: IL2RG _fw: 5′-TCACACAGCACATATTTGCCACACCCTCTG-3′; IL2RG _RV: 5′-TGCCCACATGATTGTAATGGCCAGTGG-3′; HBB _fw: 5′-CCAACTCCT AAGCCAGTGCCAGAAGAG-3′; HBB _rv: 5′-AGTCAGTGCCTATCAGAAAC CCAAGAG-3′; CCR5 _fw: 5′-GCACAGGGTGGAACAAGATGG-3′; CCR5_rv: 5′-CACCACCCCAAAGGTGACCGT-3′.

    Techniques: Modification, Plasmid Preparation, Polymerase Chain Reaction, Cleavage Assay

    Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. ( a ) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. ( b ). ( c , d amplicons ( c ) or gene addition by HR at the three loci IL2RG , HBB and CCR5 with synthetic sgRNAs ( d ). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. ( e ) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. ( f ) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. ( g ) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

    Journal: Nature biotechnology

    Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

    doi: 10.1038/nbt.3290

    Figure Lengend Snippet: Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. ( a ) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. ( b ). ( c , d amplicons ( c ) or gene addition by HR at the three loci IL2RG , HBB and CCR5 with synthetic sgRNAs ( d ). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. ( e ) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. ( f ) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. ( g ) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

    Article Snippet: PCR amplicons spanning the sgRNA genomic target sites were generated using the iProof High-Fidelity Master Mix (Bio-Rad, Hercules, CA, USA) with the following primer pairs: IL2RG _fw: 5′-TCACACAGCACATATTTGCCACACCCTCTG-3′; IL2RG _RV: 5′-TGCCCACATGATTGTAATGGCCAGTGG-3′; HBB _fw: 5′-CCAACTCCT AAGCCAGTGCCAGAAGAG-3′; HBB _rv: 5′-AGTCAGTGCCTATCAGAAAC CCAAGAG-3′; CCR5 _fw: 5′-GCACAGGGTGGAACAAGATGG-3′; CCR5_rv: 5′-CACCACCCCAAAGGTGACCGT-3′.

    Techniques: Synthesized, Modification, Sequencing, Plasmid Preparation, Positive Control, Polymerase Chain Reaction, Electroporation

    Mutation frequency by gene from results of ( a ) MALDI-TOF, n = 827, and ( b ) TruSeq Amplicon Cancer Panel, n = 792. Mutation frequency was calculated as number of variant occurrences within each gene divided by the total number of patients

    Journal: Genome Medicine

    Article Title: Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial

    doi: 10.1186/s13073-016-0364-2

    Figure Lengend Snippet: Mutation frequency by gene from results of ( a ) MALDI-TOF, n = 827, and ( b ) TruSeq Amplicon Cancer Panel, n = 792. Mutation frequency was calculated as number of variant occurrences within each gene divided by the total number of patients

    Article Snippet: Three molecular profiling assays were used over the study period: a custom multiplex genotyping panel on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass-spectrometry platform (MassARRAY, Agena Bioscience, San Diego, CA, USA) to genotype 279 mutations within 23 genes (Additional file : Table S1); the TruSeq Amplicon Cancer Panel (TSACP, Illumina) on the MiSeq sequencer (Illumina) covering regions of 48 genes (Additional file : Table S2); and the Ion AmpliSeq Cancer Panel (ASCP, ThermoFisher Scientific) on the Ion Proton sequencer (ThermoFisher Scientific) covering regions of 50 genes (Additional file : Table S3).

    Techniques: Mutagenesis, Amplification, Variant Assay

    Distribution of patients by tumor site and most actionable variant identified [ 4 ]. Cases tested with TruSeq Amplicon Cancer Panel (TSACP; n = 792) are shown in ( a ) and ( b ); cases tested by MALDI-TOF MS (n = 827) are shown in ( c ) and ( d ). a Proportion and number of variants by tumor site, TSACP. b Actionability of variants by tumor site, TSACP. c Proportion and number of variants by tumor site, MALDI-TOF. d Actionability of variants per case by tumor site, MALDI-TOF. Patients with more than one variant were counted once by their most actionable variant class. Total number of patients is indicated by value within or below each bar section . “Gyne-other” includes cervical, endometrial, fallopian tube, uterine, and vulvar

    Journal: Genome Medicine

    Article Title: Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial

    doi: 10.1186/s13073-016-0364-2

    Figure Lengend Snippet: Distribution of patients by tumor site and most actionable variant identified [ 4 ]. Cases tested with TruSeq Amplicon Cancer Panel (TSACP; n = 792) are shown in ( a ) and ( b ); cases tested by MALDI-TOF MS (n = 827) are shown in ( c ) and ( d ). a Proportion and number of variants by tumor site, TSACP. b Actionability of variants by tumor site, TSACP. c Proportion and number of variants by tumor site, MALDI-TOF. d Actionability of variants per case by tumor site, MALDI-TOF. Patients with more than one variant were counted once by their most actionable variant class. Total number of patients is indicated by value within or below each bar section . “Gyne-other” includes cervical, endometrial, fallopian tube, uterine, and vulvar

    Article Snippet: Three molecular profiling assays were used over the study period: a custom multiplex genotyping panel on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass-spectrometry platform (MassARRAY, Agena Bioscience, San Diego, CA, USA) to genotype 279 mutations within 23 genes (Additional file : Table S1); the TruSeq Amplicon Cancer Panel (TSACP, Illumina) on the MiSeq sequencer (Illumina) covering regions of 48 genes (Additional file : Table S2); and the Ion AmpliSeq Cancer Panel (ASCP, ThermoFisher Scientific) on the Ion Proton sequencer (ThermoFisher Scientific) covering regions of 50 genes (Additional file : Table S3).

    Techniques: Variant Assay, Amplification, Mass Spectrometry

    Sequence accuracy as a function of subread depth. a Accuracy of consensus and b assembly sequences. Data from all the amplicons were pooled together to evaluate the consensus calling accuracy as a function of depth of coverage of SMRT raw reads. The vertical line shows the minimum read depth of the consensus sequences used for assemblies

    Journal: BMC Bioinformatics

    Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

    doi: 10.1186/s12859-018-2293-0

    Figure Lengend Snippet: Sequence accuracy as a function of subread depth. a Accuracy of consensus and b assembly sequences. Data from all the amplicons were pooled together to evaluate the consensus calling accuracy as a function of depth of coverage of SMRT raw reads. The vertical line shows the minimum read depth of the consensus sequences used for assemblies

    Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

    Techniques: Sequencing

    Total number of accurate bootstrap assemblies per CCS sample size. At each level of the CCS read depth sample (1-40), the figure shows the total number of bootstrapped assemblies that were 100% identical to the reference sequence. This was determined for the four target regions (25 bootstrap assemblies at each of 4 loci, giving rise to a maximum of 100 on the x-axis) formed from the consensus sequences among the eight overlapping amplicons

    Journal: BMC Bioinformatics

    Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

    doi: 10.1186/s12859-018-2293-0

    Figure Lengend Snippet: Total number of accurate bootstrap assemblies per CCS sample size. At each level of the CCS read depth sample (1-40), the figure shows the total number of bootstrapped assemblies that were 100% identical to the reference sequence. This was determined for the four target regions (25 bootstrap assemblies at each of 4 loci, giving rise to a maximum of 100 on the x-axis) formed from the consensus sequences among the eight overlapping amplicons

    Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

    Techniques: Sequencing

    Graphical representation of the C3S-LAA process and pipeline. a Raw reads comprised of multiple subreads are depicted for three different amplicons [green, fuchsia and blue boxes; different shades of color are used to portray variable subread sequence qualities (darker shading portrays higher quality)]. Subreads are separated by a shared adapter sequence (grey boxes). The higher quality CCS read for each raw read is used to cluster the corresponding raw reads into CCS-based cluster groups. Error correction is performed per CCS-based cluster, producing top quality consequences sequences, followed by assembly of any overlapping consensus sequences. b A single run parameters file is used by all components of the pipeline. The grey highlighted rectangles represent two main steps of C3S-LAA. (i) Using the CCS reads generated by the SMRT analysis reads of insert protocol, C3S clusters the raw reads according to each barcode-primer pair combination, producing files of read identifiers to whitelist the corresponding raw reads. (ii) Raw read clusters are passed to Quiver to generate amplicon-specific consensus sequences, which are then passed to Minimus for sequence assembly. Rectangles with folded corners represent single files or multiple files (depicted as stacks of files) and those with rounded edges represent scripts and tools. Arrows indicates output files that are generated. Connecting lines with dots at one end depict input files, with the dot corresponding to the source data for the connected script or tool

    Journal: BMC Bioinformatics

    Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

    doi: 10.1186/s12859-018-2293-0

    Figure Lengend Snippet: Graphical representation of the C3S-LAA process and pipeline. a Raw reads comprised of multiple subreads are depicted for three different amplicons [green, fuchsia and blue boxes; different shades of color are used to portray variable subread sequence qualities (darker shading portrays higher quality)]. Subreads are separated by a shared adapter sequence (grey boxes). The higher quality CCS read for each raw read is used to cluster the corresponding raw reads into CCS-based cluster groups. Error correction is performed per CCS-based cluster, producing top quality consequences sequences, followed by assembly of any overlapping consensus sequences. b A single run parameters file is used by all components of the pipeline. The grey highlighted rectangles represent two main steps of C3S-LAA. (i) Using the CCS reads generated by the SMRT analysis reads of insert protocol, C3S clusters the raw reads according to each barcode-primer pair combination, producing files of read identifiers to whitelist the corresponding raw reads. (ii) Raw read clusters are passed to Quiver to generate amplicon-specific consensus sequences, which are then passed to Minimus for sequence assembly. Rectangles with folded corners represent single files or multiple files (depicted as stacks of files) and those with rounded edges represent scripts and tools. Arrows indicates output files that are generated. Connecting lines with dots at one end depict input files, with the dot corresponding to the source data for the connected script or tool

    Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

    Techniques: Sequencing, Generated, Amplification

    Efficient correction of OTC-deficient patient-derived primary human hepatocytes in vivo . (A) Experimental overview. Cells were engrafted into FRG mice and repopulation established by cycling on and off the drug 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione. 20 Eleven weeks later, when the mice were on day 11 of a 21-day water cycle, animals received 5×10 10 vg/mouse SaCas9-sgRNA4 and 2×10 11 vg/mouse donor vectors, 2.5×10 10 vg/mouse SaCas9-sgRNA4 and 1×10 11 vg/mouse donor vectors or 1.25×10 10 vg/mouse SaCas9-sgRNA4 and 5×10 10 vg/mouse donor vectors packaged in the NP59 capsid via intraperitoneal delivery (n = 3 per treatment group for controls and highest vector dose, and n = 4 for 2 lower vector doses). Livers were analyzed 5 weeks following vector delivery. Next-generation Illumina® sequencing was performed across the OTC locus from (B, C) genomic DNA and (D, E) cDNA isolated from FRG mice transplanted with patient-derived human hepatocytes to quantitate the HDR rates and characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. Control samples represent PCR amplicons from engrafted FRG mice that did not receive AAV vector treatment. AAV, adeno-associated virus; FRG, Fah -/- Rag2 -/- Il2rg -/ ; HDR, homology-directed repair; InDels, insertions and deletions; ITR, inverted terminal repeat; SaCas9, Staphylococcus aureus Cas9 nuclease; sgRNA, single guide RNA; rAAV, recombinant AAV.

    Journal: JHEP Reports

    Article Title: Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

    doi: 10.1016/j.jhepr.2019.100065

    Figure Lengend Snippet: Efficient correction of OTC-deficient patient-derived primary human hepatocytes in vivo . (A) Experimental overview. Cells were engrafted into FRG mice and repopulation established by cycling on and off the drug 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione. 20 Eleven weeks later, when the mice were on day 11 of a 21-day water cycle, animals received 5×10 10 vg/mouse SaCas9-sgRNA4 and 2×10 11 vg/mouse donor vectors, 2.5×10 10 vg/mouse SaCas9-sgRNA4 and 1×10 11 vg/mouse donor vectors or 1.25×10 10 vg/mouse SaCas9-sgRNA4 and 5×10 10 vg/mouse donor vectors packaged in the NP59 capsid via intraperitoneal delivery (n = 3 per treatment group for controls and highest vector dose, and n = 4 for 2 lower vector doses). Livers were analyzed 5 weeks following vector delivery. Next-generation Illumina® sequencing was performed across the OTC locus from (B, C) genomic DNA and (D, E) cDNA isolated from FRG mice transplanted with patient-derived human hepatocytes to quantitate the HDR rates and characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. Control samples represent PCR amplicons from engrafted FRG mice that did not receive AAV vector treatment. AAV, adeno-associated virus; FRG, Fah -/- Rag2 -/- Il2rg -/ ; HDR, homology-directed repair; InDels, insertions and deletions; ITR, inverted terminal repeat; SaCas9, Staphylococcus aureus Cas9 nuclease; sgRNA, single guide RNA; rAAV, recombinant AAV.

    Article Snippet: Pooled amplicons were sent to Genewiz for library preparation and MiSeq paired-read sequencing (Illumina).

    Techniques: Derivative Assay, In Vivo, Mouse Assay, Plasmid Preparation, Sequencing, Isolation, Polymerase Chain Reaction, Recombinant

    Homology-directed repair of the transposed human OTC locus in Spf ash mice . (A) Experimental overview and timing of intraperitoneal rAAV delivery for CRISPR-SaCas9-mediated HDR in Spf ash mice. Newborn animals received 5×10 10 vg transposase and 1×10 11 vg mutant minigene vectors (packaged in rAAV2/8 capsid) and 3 weeks later, 2×10 11 SaCas9/sgRNA and/or 5×10 11 donor vectors (packaged in rAAV2/rh10 capsid). Mutant minigenes were delivered (intraperitoneal) to newborn spf ash mice using the hybrid AAV/ piggyBac transposase vector system and 3 weeks later animals received genome editing vectors; n = 5 per treatment group. HDR was confirmed 2 weeks later by (B) the presence of functional OTC activity in liver sections (scale bar = 50 μm) and (C) by Sanger sequencing analysis of cloned OTC amplicons amplified using primers with binding sites outside the region of homology with the donor vector. Next-generation Illumina® sequencing was performed across the transposed OTC human minigene locus to more accurately (D) quantitate the HDR rates and (E) characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. and significance evaluated using the Mann-Whitney non-parametric test (** p

    Journal: JHEP Reports

    Article Title: Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

    doi: 10.1016/j.jhepr.2019.100065

    Figure Lengend Snippet: Homology-directed repair of the transposed human OTC locus in Spf ash mice . (A) Experimental overview and timing of intraperitoneal rAAV delivery for CRISPR-SaCas9-mediated HDR in Spf ash mice. Newborn animals received 5×10 10 vg transposase and 1×10 11 vg mutant minigene vectors (packaged in rAAV2/8 capsid) and 3 weeks later, 2×10 11 SaCas9/sgRNA and/or 5×10 11 donor vectors (packaged in rAAV2/rh10 capsid). Mutant minigenes were delivered (intraperitoneal) to newborn spf ash mice using the hybrid AAV/ piggyBac transposase vector system and 3 weeks later animals received genome editing vectors; n = 5 per treatment group. HDR was confirmed 2 weeks later by (B) the presence of functional OTC activity in liver sections (scale bar = 50 μm) and (C) by Sanger sequencing analysis of cloned OTC amplicons amplified using primers with binding sites outside the region of homology with the donor vector. Next-generation Illumina® sequencing was performed across the transposed OTC human minigene locus to more accurately (D) quantitate the HDR rates and (E) characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. and significance evaluated using the Mann-Whitney non-parametric test (** p

    Article Snippet: Pooled amplicons were sent to Genewiz for library preparation and MiSeq paired-read sequencing (Illumina).

    Techniques: Mouse Assay, CRISPR, Mutagenesis, Plasmid Preparation, Functional Assay, Activity Assay, Sequencing, Clone Assay, Amplification, Binding Assay, MANN-WHITNEY

    (A) SYBR green dissociation curve analyses of LSG-qPCR amplicons from the Stratagene Mx3000P platform. G1, L . ( V .) braziliensis , L . ( V .) guyanensis , and L . ( V .) panamensis ; G2A/B, L . ( L .) infantum/L . ( L .) chagasi and L . ( L .) tropica ; G3, L . ( L .) amazonensis

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

    doi: 10.1128/JCM.01764-16

    Figure Lengend Snippet: (A) SYBR green dissociation curve analyses of LSG-qPCR amplicons from the Stratagene Mx3000P platform. G1, L . ( V .) braziliensis , L . ( V .) guyanensis , and L . ( V .) panamensis ; G2A/B, L . ( L .) infantum/L . ( L .) chagasi and L . ( L .) tropica ; G3, L . ( L .) amazonensis

    Article Snippet: On the basis of the amplicons' Tm values observed with the Stratagene Mx3000P and ABI 7500 platforms, Leishmania spp. (as identified by conventional ITS2-PCR followed by DNA sequencing analysis) were classified into four groups, referred to here as G1, G2A, G2B, and G3 ( and ).

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction

    For reference sizes, amplicons from the LSG-qPCR and QC-qPCR were resolved on a 1.5% agarose gel. Lanes 1 and 5, 100-bp ladder size standards; lane 2, L . ( V .) panamensis ; lane 3, L . ( L .) tropica (G2B); lane 4, L . ( L .) aethiopica ; lane 6, human beta-actin

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

    doi: 10.1128/JCM.01764-16

    Figure Lengend Snippet: For reference sizes, amplicons from the LSG-qPCR and QC-qPCR were resolved on a 1.5% agarose gel. Lanes 1 and 5, 100-bp ladder size standards; lane 2, L . ( V .) panamensis ; lane 3, L . ( L .) tropica (G2B); lane 4, L . ( L .) aethiopica ; lane 6, human beta-actin

    Article Snippet: On the basis of the amplicons' Tm values observed with the Stratagene Mx3000P and ABI 7500 platforms, Leishmania spp. (as identified by conventional ITS2-PCR followed by DNA sequencing analysis) were classified into four groups, referred to here as G1, G2A, G2B, and G3 ( and ).

    Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Microbiota diversity is not regained upon direct weaning the diet-switching group onto the high-MAC diet a , Alpha-diversity as measured by Shannon index of fecal microbiota from generation 5 mice from the high-MAC diet control (control) (n=6), generation 5, diet-switching group that was weaned directly onto the high-MAC diet (Gen 5 diet switching) (n=6), and generation 4 mice from the diet switching group after weaning and maintenance on the low-MAC diet for 13 weeks and returned to the high-MAC diet for four weeks (Gen 4 diet switching) (n=5). Error bars are shown as s.e.m. and P values are from a two-tailed Student’s t-test b , Principal coordinate analysis of unweighted UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from first generation control mice on a high-MAC diet (green), fourth generation, diet-switching mice (purple), and fifth generation mice from the diet-switching lineage weaned directly onto the high-MAC diet (orange). Control is plotted as weeks post-humanization and generation 4 and 5 are plotted as age.

    Journal: Nature

    Article Title: Diet-induced extinction in the gut microbiota compounds over generations

    doi: 10.1038/nature16504

    Figure Lengend Snippet: Microbiota diversity is not regained upon direct weaning the diet-switching group onto the high-MAC diet a , Alpha-diversity as measured by Shannon index of fecal microbiota from generation 5 mice from the high-MAC diet control (control) (n=6), generation 5, diet-switching group that was weaned directly onto the high-MAC diet (Gen 5 diet switching) (n=6), and generation 4 mice from the diet switching group after weaning and maintenance on the low-MAC diet for 13 weeks and returned to the high-MAC diet for four weeks (Gen 4 diet switching) (n=5). Error bars are shown as s.e.m. and P values are from a two-tailed Student’s t-test b , Principal coordinate analysis of unweighted UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from first generation control mice on a high-MAC diet (green), fourth generation, diet-switching mice (purple), and fifth generation mice from the diet-switching lineage weaned directly onto the high-MAC diet (orange). Control is plotted as weeks post-humanization and generation 4 and 5 are plotted as age.

    Article Snippet: 16S rRNA amplicon sequencing and analysis 16S rRNA amplicons were generated for the v4 region from fecal pellets collected and the 16,878,145 Illumina generated sequencing reads were analyzed using Qiime 1.8 as described previously .

    Techniques: Mouse Assay, Two Tailed Test, Amplification

    Inefficient inter-generational transfer of taxa driven to low abundance by low dietary MACs a , Schematic of multigeneration mouse experiment. Second (n=6), third (n=6), and fourth generation mice (n=6) were weaned onto a low-MAC diet. After mice generated a litter of pups that were weaned, low-MAC diet mice were switched to the high-MAC diet for 4 weeks. A parallel group of control mice were maintained on the high-MAC diet throughout (generation 2, n=6; generation 3, n=6; generation 4, n=5). b , Microbiota diversity as measured by Shannon index observed in the microbiota of mice at five weeks old (top panel, n=6 for each group) or four weeks after shift to high-MAC diet (bottom panel, n=6 for each group) from three generations of diet switching mice (grey) or control high-MAC diet mice (black). Error bars are shown as s.e.m and P values are from two-tailed Student’s t-test. c , Principal coordinate analysis of UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from first generation mice from the control group consuming a high-MAC diet (green, n=5) or the diet switching group from generation 1 (yellow, n=5), 2 (blue, n=6), 3 (red, n=6), and 4 (purple, n=6). d , Heat map of abundance of high-confidence OTUs (number of sequencing reads, columns) from the diet switching group (top panel) and controls (bottom panel); taxonomic assignment is indicated at the top of each column (Bacteroidetes, green; Firmicutes, orange; other, grey). Each row represents an individual mouse microbiota from four weeks post-humanization (initial), while consuming the low-MAC diet (week 9, lo, shaded yellow), and four weeks after switching to the high-MAC diet (week 15, hi, shaded grey). Corresponding time points from controls are similarly shaded. N=5, 6, 6, and 6 for the diet-switching group and n=5, 6, 6, and 5 for the control group for generations one through four respectively.

    Journal: Nature

    Article Title: Diet-induced extinction in the gut microbiota compounds over generations

    doi: 10.1038/nature16504

    Figure Lengend Snippet: Inefficient inter-generational transfer of taxa driven to low abundance by low dietary MACs a , Schematic of multigeneration mouse experiment. Second (n=6), third (n=6), and fourth generation mice (n=6) were weaned onto a low-MAC diet. After mice generated a litter of pups that were weaned, low-MAC diet mice were switched to the high-MAC diet for 4 weeks. A parallel group of control mice were maintained on the high-MAC diet throughout (generation 2, n=6; generation 3, n=6; generation 4, n=5). b , Microbiota diversity as measured by Shannon index observed in the microbiota of mice at five weeks old (top panel, n=6 for each group) or four weeks after shift to high-MAC diet (bottom panel, n=6 for each group) from three generations of diet switching mice (grey) or control high-MAC diet mice (black). Error bars are shown as s.e.m and P values are from two-tailed Student’s t-test. c , Principal coordinate analysis of UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from first generation mice from the control group consuming a high-MAC diet (green, n=5) or the diet switching group from generation 1 (yellow, n=5), 2 (blue, n=6), 3 (red, n=6), and 4 (purple, n=6). d , Heat map of abundance of high-confidence OTUs (number of sequencing reads, columns) from the diet switching group (top panel) and controls (bottom panel); taxonomic assignment is indicated at the top of each column (Bacteroidetes, green; Firmicutes, orange; other, grey). Each row represents an individual mouse microbiota from four weeks post-humanization (initial), while consuming the low-MAC diet (week 9, lo, shaded yellow), and four weeks after switching to the high-MAC diet (week 15, hi, shaded grey). Corresponding time points from controls are similarly shaded. N=5, 6, 6, and 6 for the diet-switching group and n=5, 6, 6, and 5 for the control group for generations one through four respectively.

    Article Snippet: 16S rRNA amplicon sequencing and analysis 16S rRNA amplicons were generated for the v4 region from fecal pellets collected and the 16,878,145 Illumina generated sequencing reads were analyzed using Qiime 1.8 as described previously .

    Techniques: Magnetic Cell Separation, Mouse Assay, Generated, Two Tailed Test, Amplification, Sequencing

    Taxa reduction observed in low-MAC diet is largely reversible in a single generation a , Schematic of mouse experiment. Humanized mice (n=10) were maintained on a high-MAC diet for four weeks after which half of the mice were switched to a low-MAC diet for 7 weeks. These mice were then switched back to the high-MAC diet for > 4 weeks. b , Principle coordinate analysis of the UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from the diet switching mice (yellow, n=5) and control high-MAC diet mice (green, n=5). c , Distribution of OTUs fold changes for diet switching (blue, n=5) or control (red, n=5) groups comparing baseline (4 weeks post-humanization) versus week 9 (5 weeks post-low MAC diet for “diet switch” group; top panel) and baseline versus week 15 (4 weeks after return to high-MAC diet for “diet switch” group, bottom panel).

    Journal: Nature

    Article Title: Diet-induced extinction in the gut microbiota compounds over generations

    doi: 10.1038/nature16504

    Figure Lengend Snippet: Taxa reduction observed in low-MAC diet is largely reversible in a single generation a , Schematic of mouse experiment. Humanized mice (n=10) were maintained on a high-MAC diet for four weeks after which half of the mice were switched to a low-MAC diet for 7 weeks. These mice were then switched back to the high-MAC diet for > 4 weeks. b , Principle coordinate analysis of the UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from the diet switching mice (yellow, n=5) and control high-MAC diet mice (green, n=5). c , Distribution of OTUs fold changes for diet switching (blue, n=5) or control (red, n=5) groups comparing baseline (4 weeks post-humanization) versus week 9 (5 weeks post-low MAC diet for “diet switch” group; top panel) and baseline versus week 15 (4 weeks after return to high-MAC diet for “diet switch” group, bottom panel).

    Article Snippet: 16S rRNA amplicon sequencing and analysis 16S rRNA amplicons were generated for the v4 region from fecal pellets collected and the 16,878,145 Illumina generated sequencing reads were analyzed using Qiime 1.8 as described previously .

    Techniques: Mouse Assay, Amplification

    Reintroduction of lost taxa and a high-MAC diet restores microbiota diversity and composition a , Schematic of fecal transplant mouse experiment. b , Principal coordinate analysis of UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from fourth generation control mice on a high-MAC diet (green, n=6), fourth generation, diet-switching mice that received a fecal transplant (red, n=3), or did not (blue, n=3). c , Microbiota diversity as measured by Shannon index observed in the microbiota of mice that received a fecal transplant (red, n=3) or did not (blue, n=3). A green circle denotes the number of OTUs observed in fourth generation control mice consuming a high-MAC diet (n=6). Error bars are shown as s.e.m. d , Heat map of abundance of high-confidence OTUs (number of sequencing reads) from fourth generation diet-switching mice (n=3) three to 14 days after FMT (fecal microbiota transplant) and no FMT controls (n=3); taxonomic assignment is indicated at the top of each column (Bacteroidetes, green; Firmicutes, orange; other, grey). FMT donor (fourth generation control mice, n=5) and fourth generation diet-switching mice (n=5) four weeks after consuming high-MAC diet are also shown.

    Journal: Nature

    Article Title: Diet-induced extinction in the gut microbiota compounds over generations

    doi: 10.1038/nature16504

    Figure Lengend Snippet: Reintroduction of lost taxa and a high-MAC diet restores microbiota diversity and composition a , Schematic of fecal transplant mouse experiment. b , Principal coordinate analysis of UniFrac distance for 16S rRNA amplicon profiles from fecal samples collected from fourth generation control mice on a high-MAC diet (green, n=6), fourth generation, diet-switching mice that received a fecal transplant (red, n=3), or did not (blue, n=3). c , Microbiota diversity as measured by Shannon index observed in the microbiota of mice that received a fecal transplant (red, n=3) or did not (blue, n=3). A green circle denotes the number of OTUs observed in fourth generation control mice consuming a high-MAC diet (n=6). Error bars are shown as s.e.m. d , Heat map of abundance of high-confidence OTUs (number of sequencing reads) from fourth generation diet-switching mice (n=3) three to 14 days after FMT (fecal microbiota transplant) and no FMT controls (n=3); taxonomic assignment is indicated at the top of each column (Bacteroidetes, green; Firmicutes, orange; other, grey). FMT donor (fourth generation control mice, n=5) and fourth generation diet-switching mice (n=5) four weeks after consuming high-MAC diet are also shown.

    Article Snippet: 16S rRNA amplicon sequencing and analysis 16S rRNA amplicons were generated for the v4 region from fecal pellets collected and the 16,878,145 Illumina generated sequencing reads were analyzed using Qiime 1.8 as described previously .

    Techniques: Amplification, Mouse Assay, Sequencing

    Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 amplicons served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively

    Journal: Molecular Cancer

    Article Title: Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients

    doi: 10.1186/s12943-015-0381-6

    Figure Lengend Snippet: Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 amplicons served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively

    Article Snippet: Amplicons were separated by 1 % agarose gel electrophoresis and visualized with GelRed (Biotium, USA) using the UV documentation system for agarose gel (peqlab, Germany).

    Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR, Clone Assay, BrdU Incorporation Assay