nanopore sequencing adapters  (New England Biolabs)


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    NEBNext Ultra DNA Library Prep Kit for Illumina
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    NEBNext Ultra DNA Library Prep Kit for Illumina 96 rxns
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    e7370l
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    DNA Template Preparation for PCR
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    New England Biolabs nanopore sequencing adapters
    NEBNext Ultra DNA Library Prep Kit for Illumina
    NEBNext Ultra DNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/nanopore sequencing adapters/product/New England Biolabs
    Average 93 stars, based on 2476 article reviews
    Price from $9.99 to $1999.99
    nanopore sequencing adapters - by Bioz Stars, 2020-07
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    1) Product Images from "Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity"

    Article Title: Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity

    Journal: Cell

    doi: 10.1016/j.cell.2019.01.031

    AML cellular hierarchies correlate with underlying genetic alterations A . Genome plot illustrates nanopore reads for four selected FLT3 transcripts from AML419A. For each transcript, 100 reads are shown. Black arrow indicates the location of the primer used for amplification (exon 11). Base mismatches encoding A680V (exon 16; green) or N841K (exon 20; red) mutations are indicated. Base insertions representing a 24 bp ITD are indicated in exon 14 (pink). The mutations do not co-occur on the same transcripts. B-C . Diagrams show AML419 evolution inferred from co-occurrence of mutations in single cells ( B ) and VAFs from bulk DNA sequencing ( C ). The most likely model yields one subclone with an A680V mutation, a second subclone with an ITD, and a third subclone that exclusively harbors an N841K mutation. D . Diagram shows FLT3 protein domains and location of mutations. E . Heatmap shows expression of 180 signature genes for the six malignant cell types (rows) in 40 single cells from AML419A (columns). Cells were assigned to subclone A or B, or subclone C on the basis of FLT3 genotypes. F . Heatmap shows expression of 180 signature genes for the six malignant cell types (rows) in 179 AMLs profiled by bulk RNA-seq (columns). Unsupervised clustering revealed seven subsets of patients with different inferred cell type abundances (clusters A-G). G . Charts indicate chromosomal aberrations (top), mutations (middle) and FAB classifications (bottom) for AMLs in F . Correspondence between cell type compositions and genetics is evident. P -values indicate non-random distribution of events between clusters (Fisher’s exact test). n.s., not significant. H . Flow cytometry histograms show expression of the primitive cell marker CD34 in MUTZ-3 cells, four days after transduction with FLT3-WT, FLT3-D835Y, FLT3-ITD or a control gene ( luciferase ). I . Plot shows change in the percent of CD34 + cells following transduction of FLT3 variants as in H . P -values were calculated using Student’s t -test compared to CTRL (mean + SD of n = 6 transductions). * P
    Figure Legend Snippet: AML cellular hierarchies correlate with underlying genetic alterations A . Genome plot illustrates nanopore reads for four selected FLT3 transcripts from AML419A. For each transcript, 100 reads are shown. Black arrow indicates the location of the primer used for amplification (exon 11). Base mismatches encoding A680V (exon 16; green) or N841K (exon 20; red) mutations are indicated. Base insertions representing a 24 bp ITD are indicated in exon 14 (pink). The mutations do not co-occur on the same transcripts. B-C . Diagrams show AML419 evolution inferred from co-occurrence of mutations in single cells ( B ) and VAFs from bulk DNA sequencing ( C ). The most likely model yields one subclone with an A680V mutation, a second subclone with an ITD, and a third subclone that exclusively harbors an N841K mutation. D . Diagram shows FLT3 protein domains and location of mutations. E . Heatmap shows expression of 180 signature genes for the six malignant cell types (rows) in 40 single cells from AML419A (columns). Cells were assigned to subclone A or B, or subclone C on the basis of FLT3 genotypes. F . Heatmap shows expression of 180 signature genes for the six malignant cell types (rows) in 179 AMLs profiled by bulk RNA-seq (columns). Unsupervised clustering revealed seven subsets of patients with different inferred cell type abundances (clusters A-G). G . Charts indicate chromosomal aberrations (top), mutations (middle) and FAB classifications (bottom) for AMLs in F . Correspondence between cell type compositions and genetics is evident. P -values indicate non-random distribution of events between clusters (Fisher’s exact test). n.s., not significant. H . Flow cytometry histograms show expression of the primitive cell marker CD34 in MUTZ-3 cells, four days after transduction with FLT3-WT, FLT3-D835Y, FLT3-ITD or a control gene ( luciferase ). I . Plot shows change in the percent of CD34 + cells following transduction of FLT3 variants as in H . P -values were calculated using Student’s t -test compared to CTRL (mean + SD of n = 6 transductions). * P

    Techniques Used: Amplification, DNA Sequencing, Mutagenesis, Expressing, RNA Sequencing Assay, Flow Cytometry, Marker, Transduction, Luciferase

    2) Product Images from "Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing"

    Article Title: Comparative analyses of the major royal jelly protein gene cluster in three Apis species with long amplicon sequencing

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsw064

    Graphical presentation of the data analysis pipeline. 2D raw reads (1) were size-selected (minimum read length of 6.5 kb) (2) and mapped against the three reference genomes, in order to assign the reads by species and genomic target (3). Only those reads that matched our quality filters (similarity fraction: 0.6 [0.5 for adAmp3, 6 and 7], length fraction: 0.7 [0.5 for adAmp3, 6 and 7]) were included in further analyses (4). Per amplicon sixteen reads (minimum number of reads that mapped to an amplicon—amAmp6) were selected and aligned to each other independent of a reference sequence to build the nanopore-derived consensus sequence (5). Finally, the consensus sequence and the reference sequence were aligned (6). In order to correct the genomic reference sequences of the mrjp gene cluster of A. mellifera , A. florea and A. dorsata , assembly gaps (N) and local mis-assemblies were identified based on this consensus/reference sequence alignment. Assembly gaps (N) in the reference sequence were replaced with the consensus sequence and mis-assemblies were either discarded (when only present in the reference but not in the consensus sequence) or included (when only present in the consensus but not in the reference sequence).
    Figure Legend Snippet: Graphical presentation of the data analysis pipeline. 2D raw reads (1) were size-selected (minimum read length of 6.5 kb) (2) and mapped against the three reference genomes, in order to assign the reads by species and genomic target (3). Only those reads that matched our quality filters (similarity fraction: 0.6 [0.5 for adAmp3, 6 and 7], length fraction: 0.7 [0.5 for adAmp3, 6 and 7]) were included in further analyses (4). Per amplicon sixteen reads (minimum number of reads that mapped to an amplicon—amAmp6) were selected and aligned to each other independent of a reference sequence to build the nanopore-derived consensus sequence (5). Finally, the consensus sequence and the reference sequence were aligned (6). In order to correct the genomic reference sequences of the mrjp gene cluster of A. mellifera , A. florea and A. dorsata , assembly gaps (N) and local mis-assemblies were identified based on this consensus/reference sequence alignment. Assembly gaps (N) in the reference sequence were replaced with the consensus sequence and mis-assemblies were either discarded (when only present in the reference but not in the consensus sequence) or included (when only present in the consensus but not in the reference sequence).

    Techniques Used: Amplification, Sequencing, Derivative Assay

    3) Product Images from "de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer"

    Article Title: de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

    Journal: GigaScience

    doi: 10.1093/gigascience/giw018

    Cartography of the Ty transposon family. First and second tracks show, respectively, the percentage identity of the SMARTdenovo S288C assembly before and after polishing with Illumina paired-end reads using Pilon. The third track shows the 80th percentile number of contigs obtained for each strain and for all chromosomes. The remaining tracks show the density of Ty transposons or positions of the Ty1, Ty2, Ty3, Ty4, and Ty5 transposons across all the yeast strains. The red dot on the karyotype track shows the position of the rDNA cluster.
    Figure Legend Snippet: Cartography of the Ty transposon family. First and second tracks show, respectively, the percentage identity of the SMARTdenovo S288C assembly before and after polishing with Illumina paired-end reads using Pilon. The third track shows the 80th percentile number of contigs obtained for each strain and for all chromosomes. The remaining tracks show the density of Ty transposons or positions of the Ty1, Ty2, Ty3, Ty4, and Ty5 transposons across all the yeast strains. The red dot on the karyotype track shows the position of the rDNA cluster.

    Techniques Used:

    4) Product Images from "Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse"

    Article Title: Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse

    Journal: Nature medicine

    doi: 10.1038/s41591-019-0593-1

    a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.
    Figure Legend Snippet: a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining

    5) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Figure Legend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    6) Product Images from "Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis"

    Article Title: Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/ncomms10063

    Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.
    Figure Legend Snippet: Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.

    Techniques Used: Sequencing, Diffusion-based Assay, DNA Extraction, Sample Prep

    7) Product Images from "Improved data analysis for the MinION nanopore sequencer"

    Article Title: Improved data analysis for the MinION nanopore sequencer

    Journal: Nature methods

    doi: 10.1038/nmeth.3290

    Read length distributions and identity plots for M13. Read length histograms for mapped vs. unmapped reads across three replicate M13 experiments for (a) template; (b) complement; and (c) 2D reads. Most of the reads mapped to a known reference, with two distinct peaks at about 7.2 kb, corresponding to full-length M13, and 3.8 kb, corresponding to the phage lambda DNA (control fragment). Insets show the proportion of mappable vs. unmappable reads and the proportion of unmappable reads that found hits when compared against the NCBI NT database using BLAST (to check for contamination or missed homology). Read alignment identities for mappable reads using tuned LAST, realigned LAST, and EM trained LAST for (d) template; (e) complement; and (f) 2D reads.
    Figure Legend Snippet: Read length distributions and identity plots for M13. Read length histograms for mapped vs. unmapped reads across three replicate M13 experiments for (a) template; (b) complement; and (c) 2D reads. Most of the reads mapped to a known reference, with two distinct peaks at about 7.2 kb, corresponding to full-length M13, and 3.8 kb, corresponding to the phage lambda DNA (control fragment). Insets show the proportion of mappable vs. unmappable reads and the proportion of unmappable reads that found hits when compared against the NCBI NT database using BLAST (to check for contamination or missed homology). Read alignment identities for mappable reads using tuned LAST, realigned LAST, and EM trained LAST for (d) template; (e) complement; and (f) 2D reads.

    Techniques Used: Lambda DNA Preparation

    8) Product Images from "Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia"

    Article Title: Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30330-y

    Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.
    Figure Legend Snippet: Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.

    Techniques Used: Sequencing, Amplification

    9) Product Images from "Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality"

    Article Title: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005310

    Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.
    Figure Legend Snippet: Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.

    Techniques Used: Mutagenesis, Generated, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Infection, Sequencing, Amplification, Multiplex Assay

    10) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    11) Product Images from "Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing"

    Article Title: Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep29681

    Amplicon sequencing of 16s rDNA gene. ( A ) Schematic workflow to examine the composition of the mouse gut microbiota using the nanopore (MinION) and the short-read (Illumina MiSeq) sequencing. ( B ) The distribution of PHRED quality scores of short-read sequencing data and pass 2D reads of nanopore sequencing data. ( C ) Density plot for length distribution comparison of short-read sequencing data and pass 2D reads of nanopore sequencing data. Each sample is colored separately.
    Figure Legend Snippet: Amplicon sequencing of 16s rDNA gene. ( A ) Schematic workflow to examine the composition of the mouse gut microbiota using the nanopore (MinION) and the short-read (Illumina MiSeq) sequencing. ( B ) The distribution of PHRED quality scores of short-read sequencing data and pass 2D reads of nanopore sequencing data. ( C ) Density plot for length distribution comparison of short-read sequencing data and pass 2D reads of nanopore sequencing data. Each sample is colored separately.

    Techniques Used: Amplification, Sequencing, Nanopore Sequencing

    12) Product Images from "Molecular characteristics of early-stage female germ cells revealed by RNA sequencing of low-input cells and analysis of genome-wide DNA methylation"

    Article Title: Molecular characteristics of early-stage female germ cells revealed by RNA sequencing of low-input cells and analysis of genome-wide DNA methylation

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsy042

    Genome-wide DNA methylation patterns during female germline development and methylome comparison of fresh and cultured FGSCs. (A) DNA methylation levels during female germline development. (B) Correlation analysis (log-transformed corrected read count) of the fresh FGSCs and cultured FGSCs MeDIP-ChIP data sets. (C) A snapshot of the IGV genome browser showing DNA methylation (5 m C) signal at the indicated region (top track) and Stra8 locus (bottom track). (D) Methylated CGIs identified in fresh FGSCs (the purple) and cultured FGSCs (the red) show high agreement. (E) Functional annotation of methylated genes shared by fresh FGSC and cultured FGSCs (p
    Figure Legend Snippet: Genome-wide DNA methylation patterns during female germline development and methylome comparison of fresh and cultured FGSCs. (A) DNA methylation levels during female germline development. (B) Correlation analysis (log-transformed corrected read count) of the fresh FGSCs and cultured FGSCs MeDIP-ChIP data sets. (C) A snapshot of the IGV genome browser showing DNA methylation (5 m C) signal at the indicated region (top track) and Stra8 locus (bottom track). (D) Methylated CGIs identified in fresh FGSCs (the purple) and cultured FGSCs (the red) show high agreement. (E) Functional annotation of methylated genes shared by fresh FGSC and cultured FGSCs (p

    Techniques Used: Genome Wide, DNA Methylation Assay, Cell Culture, Transformation Assay, Methylated DNA Immunoprecipitation, Chromatin Immunoprecipitation, Methylation, Functional Assay

    13) Product Images from "Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse"

    Article Title: Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse

    Journal: Nature medicine

    doi: 10.1038/s41591-019-0593-1

    Mutations present in the relapse tumour are detected 10 months earlier in PV-CTCs and not in the primary tumour. a , Patient timeline from diagnosis to death (FU=follow up; PET=positron emission tomography; MR=magnetic resonance). b , Heat map showing the comparison between CNA detected in PV-CTCs or circulating epithelial cells (CECs), in primary tumour regions (R1-3), in relapse tumour (Met) and in a WBC control. Regions of loss are coloured blue, regions of gain are coloured red. Chromosomes are indicated at the top of the figure. c , Heat map showing the comparison of SNVs detected in PV-CTCs, primary tumour regions and the metastasis. Mutations are ordered according to their clonality as established by primary tumour analysis. Green dashed boxes indicate mutations that are seen in the primary tumour, but not metastasis or PV-CTCs. Blue dashed box indicates the overlap between mutations considered metastatic private by primary tumour analysis and PV-CTCs. No mutations were found in the three CECs and two WBCs. d, Evolutionary tree encompassing tumour and PV-CTCs: the relationships between identified subclones is depicted, with size of circle reflecting the number of mutations in each subclone relative to largest. Length of lines connecting tumor subclones does not carry information. The beehive plots indicate the subclonal architecture of each tumour region, with 100 representative cells shown for each region and the nested colours corresponding to the ancestry of each cell. e, Heat map showing PV-CTC private mutations that are detected in primary tumour, metastasis and cfDNA following targeted deep sequencing.
    Figure Legend Snippet: Mutations present in the relapse tumour are detected 10 months earlier in PV-CTCs and not in the primary tumour. a , Patient timeline from diagnosis to death (FU=follow up; PET=positron emission tomography; MR=magnetic resonance). b , Heat map showing the comparison between CNA detected in PV-CTCs or circulating epithelial cells (CECs), in primary tumour regions (R1-3), in relapse tumour (Met) and in a WBC control. Regions of loss are coloured blue, regions of gain are coloured red. Chromosomes are indicated at the top of the figure. c , Heat map showing the comparison of SNVs detected in PV-CTCs, primary tumour regions and the metastasis. Mutations are ordered according to their clonality as established by primary tumour analysis. Green dashed boxes indicate mutations that are seen in the primary tumour, but not metastasis or PV-CTCs. Blue dashed box indicates the overlap between mutations considered metastatic private by primary tumour analysis and PV-CTCs. No mutations were found in the three CECs and two WBCs. d, Evolutionary tree encompassing tumour and PV-CTCs: the relationships between identified subclones is depicted, with size of circle reflecting the number of mutations in each subclone relative to largest. Length of lines connecting tumor subclones does not carry information. The beehive plots indicate the subclonal architecture of each tumour region, with 100 representative cells shown for each region and the nested colours corresponding to the ancestry of each cell. e, Heat map showing PV-CTC private mutations that are detected in primary tumour, metastasis and cfDNA following targeted deep sequencing.

    Techniques Used: Positron Emission Tomography, Sequencing

    Heat map showing the comparison of SNVs detected in primary tumour regions, metastasis, PV-CTCs, CECs, WBCs, and cfDNA samples (cfDNA pre-surgery was isolated from peripheral blood, cfDNA surgery was isolated from the pulmonary vein and cfDNA relapse was isolated at the time of relapse). Mutations are ordered according to their clonality established by primary tumour analysis.
    Figure Legend Snippet: Heat map showing the comparison of SNVs detected in primary tumour regions, metastasis, PV-CTCs, CECs, WBCs, and cfDNA samples (cfDNA pre-surgery was isolated from peripheral blood, cfDNA surgery was isolated from the pulmonary vein and cfDNA relapse was isolated at the time of relapse). Mutations are ordered according to their clonality established by primary tumour analysis.

    Techniques Used: Isolation

    14) Product Images from "Forensic SNP Genotyping using Nanopore MinION Sequencing"

    Article Title: Forensic SNP Genotyping using Nanopore MinION Sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep41759

    ( A ) Length profile (bp) of concatenated amplicons as measured with an Agilent High-Sensitivity DNA chip; internal marker at 35 bp and 10380 bp. ( B ) Read length (bp) histogram of the high quality two-directional (2D) nanopore reads.
    Figure Legend Snippet: ( A ) Length profile (bp) of concatenated amplicons as measured with an Agilent High-Sensitivity DNA chip; internal marker at 35 bp and 10380 bp. ( B ) Read length (bp) histogram of the high quality two-directional (2D) nanopore reads.

    Techniques Used: Chromatin Immunoprecipitation, Marker

    15) Product Images from "Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse"

    Article Title: Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse

    Journal: Nature medicine

    doi: 10.1038/s41591-019-0593-1

    a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.
    Figure Legend Snippet: a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining

    Mutations present in the relapse tumour are detected 10 months earlier in PV-CTCs and not in the primary tumour. a , Patient timeline from diagnosis to death (FU=follow up; PET=positron emission tomography; MR=magnetic resonance). b , Heat map showing the comparison between CNA detected in PV-CTCs or circulating epithelial cells (CECs), in primary tumour regions (R1-3), in relapse tumour (Met) and in a WBC control. Regions of loss are coloured blue, regions of gain are coloured red. Chromosomes are indicated at the top of the figure. c , Heat map showing the comparison of SNVs detected in PV-CTCs, primary tumour regions and the metastasis. Mutations are ordered according to their clonality as established by primary tumour analysis. Green dashed boxes indicate mutations that are seen in the primary tumour, but not metastasis or PV-CTCs. Blue dashed box indicates the overlap between mutations considered metastatic private by primary tumour analysis and PV-CTCs. No mutations were found in the three CECs and two WBCs. d, Evolutionary tree encompassing tumour and PV-CTCs: the relationships between identified subclones is depicted, with size of circle reflecting the number of mutations in each subclone relative to largest. Length of lines connecting tumor subclones does not carry information. The beehive plots indicate the subclonal architecture of each tumour region, with 100 representative cells shown for each region and the nested colours corresponding to the ancestry of each cell. e, Heat map showing PV-CTC private mutations that are detected in primary tumour, metastasis and cfDNA following targeted deep sequencing.
    Figure Legend Snippet: Mutations present in the relapse tumour are detected 10 months earlier in PV-CTCs and not in the primary tumour. a , Patient timeline from diagnosis to death (FU=follow up; PET=positron emission tomography; MR=magnetic resonance). b , Heat map showing the comparison between CNA detected in PV-CTCs or circulating epithelial cells (CECs), in primary tumour regions (R1-3), in relapse tumour (Met) and in a WBC control. Regions of loss are coloured blue, regions of gain are coloured red. Chromosomes are indicated at the top of the figure. c , Heat map showing the comparison of SNVs detected in PV-CTCs, primary tumour regions and the metastasis. Mutations are ordered according to their clonality as established by primary tumour analysis. Green dashed boxes indicate mutations that are seen in the primary tumour, but not metastasis or PV-CTCs. Blue dashed box indicates the overlap between mutations considered metastatic private by primary tumour analysis and PV-CTCs. No mutations were found in the three CECs and two WBCs. d, Evolutionary tree encompassing tumour and PV-CTCs: the relationships between identified subclones is depicted, with size of circle reflecting the number of mutations in each subclone relative to largest. Length of lines connecting tumor subclones does not carry information. The beehive plots indicate the subclonal architecture of each tumour region, with 100 representative cells shown for each region and the nested colours corresponding to the ancestry of each cell. e, Heat map showing PV-CTC private mutations that are detected in primary tumour, metastasis and cfDNA following targeted deep sequencing.

    Techniques Used: Positron Emission Tomography, Sequencing

    Heat map showing the comparison of SNVs detected in primary tumour regions, metastasis, PV-CTCs, CECs, WBCs, and cfDNA samples (cfDNA pre-surgery was isolated from peripheral blood, cfDNA surgery was isolated from the pulmonary vein and cfDNA relapse was isolated at the time of relapse). Mutations are ordered according to their clonality established by primary tumour analysis.
    Figure Legend Snippet: Heat map showing the comparison of SNVs detected in primary tumour regions, metastasis, PV-CTCs, CECs, WBCs, and cfDNA samples (cfDNA pre-surgery was isolated from peripheral blood, cfDNA surgery was isolated from the pulmonary vein and cfDNA relapse was isolated at the time of relapse). Mutations are ordered according to their clonality established by primary tumour analysis.

    Techniques Used: Isolation

    16) Product Images from "Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression"

    Article Title: Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression

    Journal: bioRxiv

    doi: 10.1101/484956

    Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).
    Figure Legend Snippet: Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).

    Techniques Used: Generated, Produced

    Analysis of categorized genes expressed in differentiating CGN. a Outline of the protocol used for the expression analysis in culture. RNA samples used for mRNA-seq analysis were prepared at the time points indicated. b Two-dimensional logarithmic plot of FPKM values obtained from mRNA-seq experiment. Fold-induction during differentiation (D5/D1) was plotted against fold-susceptibility to topo II inhibitor (D5/D5+). exRefSeq genes were divided into 9 groups by the dotted boarder lines that indicate 1.5- or 0.66-fold. c A pie graph and a breakdown list for the constituent expression groups. Group ‘D’ represents unexpressed genes. d Shared GO term analysis. Percentages of significant terms shared between other groups are plotted in the bar graph. Term numbers for hundred percent are shown in the bar. e Shared GO term analysis conducted with expression groups that are sorted into ‘long’ and ‘short’ subgroups. The threshold length was determined as in Fig. S11. Shared term numbers between sorted and unsorted were expressed as percentages of unsorted groups. f Correlation of Ts2 toposites to topo IIβ dependency. Ts2 toposites were counted either within the gene body (top) or within EU (middle). By pairwise homology search with EMBOSS Water algorithm, Ts2 pairs with high SW score (1000
    Figure Legend Snippet: Analysis of categorized genes expressed in differentiating CGN. a Outline of the protocol used for the expression analysis in culture. RNA samples used for mRNA-seq analysis were prepared at the time points indicated. b Two-dimensional logarithmic plot of FPKM values obtained from mRNA-seq experiment. Fold-induction during differentiation (D5/D1) was plotted against fold-susceptibility to topo II inhibitor (D5/D5+). exRefSeq genes were divided into 9 groups by the dotted boarder lines that indicate 1.5- or 0.66-fold. c A pie graph and a breakdown list for the constituent expression groups. Group ‘D’ represents unexpressed genes. d Shared GO term analysis. Percentages of significant terms shared between other groups are plotted in the bar graph. Term numbers for hundred percent are shown in the bar. e Shared GO term analysis conducted with expression groups that are sorted into ‘long’ and ‘short’ subgroups. The threshold length was determined as in Fig. S11. Shared term numbers between sorted and unsorted were expressed as percentages of unsorted groups. f Correlation of Ts2 toposites to topo IIβ dependency. Ts2 toposites were counted either within the gene body (top) or within EU (middle). By pairwise homology search with EMBOSS Water algorithm, Ts2 pairs with high SW score (1000

    Techniques Used: Expressing

    Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.
    Figure Legend Snippet: Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.

    Techniques Used: In Vitro, Staining, MANN-WHITNEY, Proximity Ligation Assay, Fluorescence In Situ Hybridization

    Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.
    Figure Legend Snippet: Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.

    Techniques Used: Isolation, Magnetic Beads, Fractionation, Ligation

    Analysis of PSP chimeras. a Size distribution curves. b Location of PSP chimeras in different genomic regions as defined in Fig. 2d . c Density plot of PSP chimera distribution. d Difference in the genomic distribution of Ts3/PSP in two groups of chimera size. Short Ts3/PSP is accounted for by satellite I repeat in intergenic region (filled area). e Conditions for dividing Ts1/PSP into two groups on the basis of association with TSS zone (TSS +/- 2 kb). Sequence reads on chimera ends are designated by rectangles. f A box plot for length distribution of Ts1/PSP. Horizontal bars indicate median length. TSS-associated Ts1/PSP is significantly longer than non-associated ones. g Aggregation plot. CpG island, SP120 site, Ts1 toposite, and Ts1/PSP chimera overlapping with TSS zone (shadowed) were aggregated. Annotation data for RefSeq genes and CpG island were downloaded from the UCSC genome browser site. The right scale applies only to Ts1/PSP. h A model for the relationship between topo IIβ action site and SP120 binding site in the vicinity of TSS. This is based on the aggregation data shown in ‘g’. A plectonemic loop with positive turn (PSP loop) is most likely to be formed in the region.
    Figure Legend Snippet: Analysis of PSP chimeras. a Size distribution curves. b Location of PSP chimeras in different genomic regions as defined in Fig. 2d . c Density plot of PSP chimera distribution. d Difference in the genomic distribution of Ts3/PSP in two groups of chimera size. Short Ts3/PSP is accounted for by satellite I repeat in intergenic region (filled area). e Conditions for dividing Ts1/PSP into two groups on the basis of association with TSS zone (TSS +/- 2 kb). Sequence reads on chimera ends are designated by rectangles. f A box plot for length distribution of Ts1/PSP. Horizontal bars indicate median length. TSS-associated Ts1/PSP is significantly longer than non-associated ones. g Aggregation plot. CpG island, SP120 site, Ts1 toposite, and Ts1/PSP chimera overlapping with TSS zone (shadowed) were aggregated. Annotation data for RefSeq genes and CpG island were downloaded from the UCSC genome browser site. The right scale applies only to Ts1/PSP. h A model for the relationship between topo IIβ action site and SP120 binding site in the vicinity of TSS. This is based on the aggregation data shown in ‘g’. A plectonemic loop with positive turn (PSP loop) is most likely to be formed in the region.

    Techniques Used: Sequencing, Binding Assay

    17) Product Images from "Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis"

    Article Title: Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/ncomms10063

    Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.
    Figure Legend Snippet: Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.

    Techniques Used: Sequencing, Diffusion-based Assay, DNA Extraction, Sample Prep

    18) Product Images from "A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing"

    Article Title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep37563

    Process for efficient, zero-background, cloning of uniquely barcoded dA-tailed fragment libraries. (a) To achieve ligation of one unique fragment into each plasmid backbone, a relatively small cloning vector (5.7 kb) was generated containing a ccdB toxin gene flanked by two specifically tailored XcmI restriction enzyme cleavage sites. (b) The sequence is tailored so that XcmI enzyme digestion leaves a single 5′ dT-overhang on both open ends of the backbone generating a T-vector where the dA-tailed DNA fragments can be efficiently ligated without directional bias. (c) The cloning vector can have a flexible design with any 5′ and 3′ domain modulated by the inserted fragment. To allow for unique barcoding of each fragment together with the surrounding 5′ and 3′ domains, the region of interest in the cloning vector is then exposed to a two-step PCR amplification where a 5′ AttB1 site is inserted in the first amplification (20 cycles amplification). (d) The barcode together with the AttB2 site are added through a PCR with only a single elongation step, ensuring that each unique barcode is utilized only once and is not transferred to other amplicons due to PCR template switching. (e) The library of uniquely barcoded PCR amplicons is then inserted into the viral vector backbone using the “Gateway” BP clonase recombination reaction. (f) Chemically or electro-competent bacteria are then transformed and a small fraction of the transformation reaction plated for a rough estimation of total number of colonies. Generation of empty backbones ( i.e., missing a genomic fragment) is kept to an absolute minimal as any such plasmid would contain the ccdB toxin gene providing negative selection pressure.
    Figure Legend Snippet: Process for efficient, zero-background, cloning of uniquely barcoded dA-tailed fragment libraries. (a) To achieve ligation of one unique fragment into each plasmid backbone, a relatively small cloning vector (5.7 kb) was generated containing a ccdB toxin gene flanked by two specifically tailored XcmI restriction enzyme cleavage sites. (b) The sequence is tailored so that XcmI enzyme digestion leaves a single 5′ dT-overhang on both open ends of the backbone generating a T-vector where the dA-tailed DNA fragments can be efficiently ligated without directional bias. (c) The cloning vector can have a flexible design with any 5′ and 3′ domain modulated by the inserted fragment. To allow for unique barcoding of each fragment together with the surrounding 5′ and 3′ domains, the region of interest in the cloning vector is then exposed to a two-step PCR amplification where a 5′ AttB1 site is inserted in the first amplification (20 cycles amplification). (d) The barcode together with the AttB2 site are added through a PCR with only a single elongation step, ensuring that each unique barcode is utilized only once and is not transferred to other amplicons due to PCR template switching. (e) The library of uniquely barcoded PCR amplicons is then inserted into the viral vector backbone using the “Gateway” BP clonase recombination reaction. (f) Chemically or electro-competent bacteria are then transformed and a small fraction of the transformation reaction plated for a rough estimation of total number of colonies. Generation of empty backbones ( i.e., missing a genomic fragment) is kept to an absolute minimal as any such plasmid would contain the ccdB toxin gene providing negative selection pressure.

    Techniques Used: Clone Assay, Ligation, Plasmid Preparation, Generated, Sequencing, Polymerase Chain Reaction, Amplification, Transformation Assay, Selection

    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
    Figure Legend Snippet: Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.

    Techniques Used: Sequencing, Ligation, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Mutagenesis, Binding Assay, Generated

    Fragmentation of genetic sequences for insertion in barcode labelled plasmids. (a) A novel version of the Pfu-based proof-reading polymerase “Phusion U” allows for insertion of dUTP during PCR amplification. However, efficiency of amplification is reduced with increasing dUTP/dTTP fraction as exemplified through amplification of a genomic sequence (1.1 kb long) utilized in library 1. (b) The percentage dUTP used in the PCR reaction is determining the distribution of fragment sizes after nicking by Uracil-DNA-Glycosylase (UDG) and NaOH single strand breakage. (c) As the cleavage sites are strictly sequence dependent and statistically predictable based on dUTP fraction and insertion frequency it can be simulated using the NExTProg 1.0 software. Here this was modelled using the sequence for library 1. (d) The dUTP/UDG fragmentation protocol was applied to three amplicons with different sequences but similar length and GC content (49–52%), aimed for inclusion in library 3, and one shorter amplicon, with 10% dUTP. (e,f) The fragments from the first lane were further analysed by higher accuracy electrophoretic analysis (Bioanalyzer) and (g) Illumina sequencing was performed after insertion into a plasmid backbone (library 3).
    Figure Legend Snippet: Fragmentation of genetic sequences for insertion in barcode labelled plasmids. (a) A novel version of the Pfu-based proof-reading polymerase “Phusion U” allows for insertion of dUTP during PCR amplification. However, efficiency of amplification is reduced with increasing dUTP/dTTP fraction as exemplified through amplification of a genomic sequence (1.1 kb long) utilized in library 1. (b) The percentage dUTP used in the PCR reaction is determining the distribution of fragment sizes after nicking by Uracil-DNA-Glycosylase (UDG) and NaOH single strand breakage. (c) As the cleavage sites are strictly sequence dependent and statistically predictable based on dUTP fraction and insertion frequency it can be simulated using the NExTProg 1.0 software. Here this was modelled using the sequence for library 1. (d) The dUTP/UDG fragmentation protocol was applied to three amplicons with different sequences but similar length and GC content (49–52%), aimed for inclusion in library 3, and one shorter amplicon, with 10% dUTP. (e,f) The fragments from the first lane were further analysed by higher accuracy electrophoretic analysis (Bioanalyzer) and (g) Illumina sequencing was performed after insertion into a plasmid backbone (library 3).

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Software, Plasmid Preparation

    19) Product Images from "Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase"

    Article Title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase

    Journal: Genome Research

    doi: 10.1101/gr.196857.115

    Quantitative BrdU-IP-seq analysis. ( A ) Scheme of the method. BrdU-labeled genomic DNA from each sample is barcoded by end-ligation of Illumina-compatible linkers. Samples are pooled, a small fraction of this pool is set aside as “Input,” and the remainder is subjected to immunoprecipitation (IP) with anti-BrdU antibody. The IP and Input samples are PCR-amplified with indexed primers and sequenced. IP sample reads are normalized against Input sample reads. ( B ) Validation of the method. A BrdU-labeled genomic DNA sample was split in two and each aliquot was uniquely barcoded. The distinctly barcoded samples were pooled at a ratio of 1:10 (color-keyed) and processed as above. IP results are shown raw and with normalization against the Input. ( C ) Comparison to CPM and Quantile normalization (Q-norm). JPy88 (WT) cells were synchronized in G1 phase with α-factor, released into S phase, and aliquots were incubated with BrdU for the indicated time intervals and harvested. The samples were processed as described for qBrdU-seq in A , and the IP sequence reads were analyzed by qBrdU-seq, CPM, or Quantile normalization and plotted as overlays of the time points.
    Figure Legend Snippet: Quantitative BrdU-IP-seq analysis. ( A ) Scheme of the method. BrdU-labeled genomic DNA from each sample is barcoded by end-ligation of Illumina-compatible linkers. Samples are pooled, a small fraction of this pool is set aside as “Input,” and the remainder is subjected to immunoprecipitation (IP) with anti-BrdU antibody. The IP and Input samples are PCR-amplified with indexed primers and sequenced. IP sample reads are normalized against Input sample reads. ( B ) Validation of the method. A BrdU-labeled genomic DNA sample was split in two and each aliquot was uniquely barcoded. The distinctly barcoded samples were pooled at a ratio of 1:10 (color-keyed) and processed as above. IP results are shown raw and with normalization against the Input. ( C ) Comparison to CPM and Quantile normalization (Q-norm). JPy88 (WT) cells were synchronized in G1 phase with α-factor, released into S phase, and aliquots were incubated with BrdU for the indicated time intervals and harvested. The samples were processed as described for qBrdU-seq in A , and the IP sequence reads were analyzed by qBrdU-seq, CPM, or Quantile normalization and plotted as overlays of the time points.

    Techniques Used: Labeling, Ligation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Incubation, Sequencing

    20) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    21) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    22) Product Images from "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform"

    Article Title: Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200087

    Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).
    Figure Legend Snippet: Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before ( wei and williams 2016 ); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands > 500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands > 500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

    Techniques Used: Ligation, Titration, Purification, Concentration Assay, Incubation

    23) Product Images from "T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids"

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    Journal: Scientific Reports

    doi: 10.1038/srep40767

    Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification, Generated

    Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction

    TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Amplification

    Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

    24) Product Images from "Generation of a transgenic ORFeome library in Drosophila"

    Article Title: Generation of a transgenic ORFeome library in Drosophila

    Journal: Nature protocols

    doi: 10.1038/nprot.2014.105

    ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.
    Figure Legend Snippet: ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.

    Techniques Used: Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Next-Generation Sequencing, Plasmid Preparation, Molecular Weight, Multiplexing

    25) Product Images from "Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression"

    Article Title: Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression

    Journal: bioRxiv

    doi: 10.1101/484956

    Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).
    Figure Legend Snippet: Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).

    Techniques Used: Generated, Produced

    Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.
    Figure Legend Snippet: Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.

    Techniques Used: In Vitro, Staining, MANN-WHITNEY, Proximity Ligation Assay, Fluorescence In Situ Hybridization

    Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.
    Figure Legend Snippet: Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.

    Techniques Used: Isolation, Magnetic Beads, Fractionation, Ligation

    26) Product Images from "Human transposon insertion profiling by sequencing (TIPseq) to map LINE-1 insertions in single cells"

    Article Title: Human transposon insertion profiling by sequencing (TIPseq) to map LINE-1 insertions in single cells

    Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

    doi: 10.1098/rstb.2019.0335

    Total number of insertions predicted. ( a ) Including all predictions. ( b ) Including only known L1Hs insertion sites including the reference L1Hs and published polymorphic L1Hs. Black dashed line, bulk DNA regular TIPseq; red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T). Circles, WGA using random hexamers only (R); squares, WGA using random hexamers and L1 primer (RL). Arrows indicate the MDA sample included in the vectorette PCR (both MDA-D and MDA-T) and next-generation sequencing stages that had less than perfect QC (corresponds to the same sample indicated in figure 2 ).
    Figure Legend Snippet: Total number of insertions predicted. ( a ) Including all predictions. ( b ) Including only known L1Hs insertion sites including the reference L1Hs and published polymorphic L1Hs. Black dashed line, bulk DNA regular TIPseq; red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T). Circles, WGA using random hexamers only (R); squares, WGA using random hexamers and L1 primer (RL). Arrows indicate the MDA sample included in the vectorette PCR (both MDA-D and MDA-T) and next-generation sequencing stages that had less than perfect QC (corresponds to the same sample indicated in figure 2 ).

    Techniques Used: Multiple Displacement Amplification, Whole Genome Amplification, Ligation, Multiple Annealing and Looping–Based Amplification Cycles, Polymerase Chain Reaction, Next-Generation Sequencing

    Comparison to ‘gold standard’ known insertions. Sensitivity and PPV when comparing single-cell TIPseq to a set of known GM12878 insertions with intact primer binding sites. ( a ) Sensitivity and PPV for all experiments, including all insertions and using a probability cut-off of 0.9. ( b ) As ( a ), but including only insertions that pass our three filters. Diamond, bulk DNA TIPseq; circle, WGA using random hexamers only (R); square, whole-genome amplification using random hexamers and L1 primer (RL). Arrows indicate the MDA sample included in the vectorette PCR (both MDA-D and MDA-T) and next-generation sequencing stages that had less than perfect QC (corresponds to the same sample indicated in figure 2 ). ( c–h ) Sensitivity–PPV curves for each single-cell TIPseq experiment as the probability cut-off is varied from 0 to 1. Black lines, bulk DNA TIPseq; red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T).
    Figure Legend Snippet: Comparison to ‘gold standard’ known insertions. Sensitivity and PPV when comparing single-cell TIPseq to a set of known GM12878 insertions with intact primer binding sites. ( a ) Sensitivity and PPV for all experiments, including all insertions and using a probability cut-off of 0.9. ( b ) As ( a ), but including only insertions that pass our three filters. Diamond, bulk DNA TIPseq; circle, WGA using random hexamers only (R); square, whole-genome amplification using random hexamers and L1 primer (RL). Arrows indicate the MDA sample included in the vectorette PCR (both MDA-D and MDA-T) and next-generation sequencing stages that had less than perfect QC (corresponds to the same sample indicated in figure 2 ). ( c–h ) Sensitivity–PPV curves for each single-cell TIPseq experiment as the probability cut-off is varied from 0 to 1. Black lines, bulk DNA TIPseq; red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T).

    Techniques Used: Binding Assay, Whole Genome Amplification, Multiple Displacement Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Ligation, Multiple Annealing and Looping–Based Amplification Cycles

    Quality control check following WGA. Pink, MDA WGA with or without L1 primer (MDA); light blue, MALBAC WGA with or without L1 primer (MALBAC). Circles, WGA using random hexamers only (R); squares, WGA using random hexamers and L1 primer (RL). Filled shapes, samples picked for following TIPseq; open shapes, samples that were not selected for the following TIPseq. An arrows indicates an MDA sample that is included in the following vectorette PCR and next-generation sequencing, but has only 11/12 regions amplified by qPCR, while other MDA samples have 12/12 regions amplified. * t -test, p = 0.0279.
    Figure Legend Snippet: Quality control check following WGA. Pink, MDA WGA with or without L1 primer (MDA); light blue, MALBAC WGA with or without L1 primer (MALBAC). Circles, WGA using random hexamers only (R); squares, WGA using random hexamers and L1 primer (RL). Filled shapes, samples picked for following TIPseq; open shapes, samples that were not selected for the following TIPseq. An arrows indicates an MDA sample that is included in the following vectorette PCR and next-generation sequencing, but has only 11/12 regions amplified by qPCR, while other MDA samples have 12/12 regions amplified. * t -test, p = 0.0279.

    Techniques Used: Whole Genome Amplification, Multiple Displacement Amplification, Multiple Annealing and Looping–Based Amplification Cycles, Polymerase Chain Reaction, Next-Generation Sequencing, Amplification, Real-time Polymerase Chain Reaction

    Overview of single-cell TIPseq workflows. The single-cell TIPseq procedure consists of five steps: cell sorting, WGA, a quality control check, vectorette PCR for L1Hs insertion site amplification and next-generation sequencing. Pink, MDA WGA with or without L1 primer (MDA); light blue, MALBAC WGA with or without L1 primer (MALBAC); red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T). Circles represent WGA using random hexamers only (R); squares represent WGA using random hexamers and the L1 primer (RL).
    Figure Legend Snippet: Overview of single-cell TIPseq workflows. The single-cell TIPseq procedure consists of five steps: cell sorting, WGA, a quality control check, vectorette PCR for L1Hs insertion site amplification and next-generation sequencing. Pink, MDA WGA with or without L1 primer (MDA); light blue, MALBAC WGA with or without L1 primer (MALBAC); red, MDA WGA followed by restriction enzyme digestion and ligation with vectorette adaptors (MDA-D); orange, MDA WGA followed by end repair, dA tailing and ligation with dT vectorette adaptor (MDA-T); dark blue, MALBAC WGA followed by ligation with dT vectorette adaptor (MALBAC-T). Circles represent WGA using random hexamers only (R); squares represent WGA using random hexamers and the L1 primer (RL).

    Techniques Used: FACS, Whole Genome Amplification, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Multiple Displacement Amplification, Multiple Annealing and Looping–Based Amplification Cycles, Ligation

    27) Product Images from "Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse"

    Article Title: Pulmonary venous circulating tumour cell dissemination before tumour resection and disease relapse

    Journal: Nature medicine

    doi: 10.1038/s41591-019-0593-1

    a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.
    Figure Legend Snippet: a, ). b , Table showing cases of relapse among the patients with single PV-CTCs isolated. c , Agarose gel showing results of a QC–PCR assay used to determine the genome integrity of each sample. 0–4 bands determine the overall DNA integrity of each sample. DEPArray images of corresponding PV-CTC (cytokeratin (CK)+ stained green, CD45+ stained blue, DAPI+ stained purple) are shown above. d , Examples of copy number profiles detected in single PV-CTCs, CECs and WBC control. Blue and red indicate regions of copy number loss and gain respectively.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining

    28) Product Images from "Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia"

    Article Title: Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30330-y

    Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.
    Figure Legend Snippet: Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.

    Techniques Used: Sequencing, Amplification

    29) Product Images from "Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia"

    Article Title: Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30330-y

    Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.
    Figure Legend Snippet: Boxplot of sequencing depth data and amplicons size (bp). The range of read depth was more uniform for longer amplicons and inversely related to the amplicon size, the smaller amplicons showing a higher sequencing depth.

    Techniques Used: Sequencing, Amplification

    30) Product Images from "T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids"

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    Journal: Scientific Reports

    doi: 10.1038/srep40767

    Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR to Illumina’s PCR method using serial dilutions of plasma cfDNA sample. Serial dilutions (5 ng–0.01 pg) of a plasma cfDNA sample isolated from a healthy female ( BBC ) was prepared and the cfDNA is amplified using either Illumina’s PCR or TOP-PCR. TOP panel: profiles generated by Illumina’s PCR method. Lower panel: profiles generated by TOP-PCR. Notice that the RFU values are no longer accurate because of figure overlay. PCR cycle numbers: 30 for 5–0.5 ng; 40 for 0.05 ng–0.01 pg. Size markers: 1 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification, Generated

    Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Test of TOP-PCR reproducibility and cfDNA consistency. Two blood samples were separately collected from a healthy male ( YFH ) on June 30, 2015 and October 28, 2015. Plasmas were prepared right after the blood collections and stored at −80 °C. Samples of cfDNA were extracted right before TOP-PCR reactions conducted on October 28, 2015. Blue, plasma stock prepared on June 30, 2015; red and black, plasma stock prepared on October 1, 2015. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction

    TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: TOP-PCR amplification of saliva and urine cfDNA. ( a ) Size profile comparison between the original and TOP-PCR amplified normal saliva DNA samples. The saliva cfDNA from a healthy male individual (YFH) was amplified by TOP-PCR and displayed in parallel with the original. (Black, 5 ng of original; blue, 1 ng of TOP-PCR product). ( b ) Comparison between the original and TOP-PCR amplified normal urine cfDNA samples. Urine sample from the same healthy male ( a ) was tested. (Black, original urine DNA; blue, 40-cycle TOP-PCR amplification of 0.1 ng of the original. 5 ng each was displayed by Fragment Analyzer. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCR with Illumina’s PCR method using low amount of DNA as the input. ( a ) One micro-liter of original ovarian cancer plasma cfDNA sample with unknown concentration. ( b ) Same DNA sample but after 50 cycles of amplification by TOP-PCR. ( c ) Same DNA sample but after 50 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay, Amplification

    Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.
    Figure Legend Snippet: Comparison of TOP-PCT with Illumina’s PCR method using 20 ng of DNA. ( a ) One nano-gram original plasma cfDNA sample isolated from a healthy female (BBC). ( b ) Same DNA sample but after 20 cycles of amplification using TOP-PCR. ( c ) Same DNA sample but after 20 cycles of amplification using Illumina’s protocol. Size markers: 35 bp and 6000 bp peaks. Size of two HAs added to each DNA fragment: ~22 bp.

    Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

    31) Product Images from "Real-time, portable genome sequencing for Ebola surveillance"

    Article Title: Real-time, portable genome sequencing for Ebola surveillance

    Journal: Nature

    doi: 10.1038/nature16996

    Primer schemes employed during the study We designed PCR primers to generate amplicons that would span the EBOV genome. We initially designed 38 primer pairs which were used in the initial validation study and which cover > 98% of the EBOV genome ( Panel A ). During in-field sequencing we used a 19 reaction scheme or 11 reaction scheme which generated longer products. The predicted amplicon products are shown with forward primers and reverse primers indicated by green bars on the forward and reverse strand respectively, scaled according to the EBOV virus coordinates. The amplicon product sizes expected are shown for the 19 reaction scheme ( Panel B ) and the 11 reaction scheme ( Panel C ). No amplicon covers the extreme 3′ region of the genome. The last primer pair, 38_R, ends at position 18578, 381 bases away from the end of the virus genome. The primer diagram was created with Biopython 33 .
    Figure Legend Snippet: Primer schemes employed during the study We designed PCR primers to generate amplicons that would span the EBOV genome. We initially designed 38 primer pairs which were used in the initial validation study and which cover > 98% of the EBOV genome ( Panel A ). During in-field sequencing we used a 19 reaction scheme or 11 reaction scheme which generated longer products. The predicted amplicon products are shown with forward primers and reverse primers indicated by green bars on the forward and reverse strand respectively, scaled according to the EBOV virus coordinates. The amplicon product sizes expected are shown for the 19 reaction scheme ( Panel B ) and the 11 reaction scheme ( Panel C ). No amplicon covers the extreme 3′ region of the genome. The last primer pair, 38_R, ends at position 18578, 381 bases away from the end of the virus genome. The primer diagram was created with Biopython 33 .

    Techniques Used: Polymerase Chain Reaction, Sequencing, Generated, Amplification

    32) Product Images from "Real-time, portable genome sequencing for Ebola surveillance"

    Article Title: Real-time, portable genome sequencing for Ebola surveillance

    Journal: Nature

    doi: 10.1038/nature16996

    Primer schemes employed during the study We designed PCR primers to generate amplicons that would span the EBOV genome. We initially designed 38 primer pairs which were used in the initial validation study and which cover > 98% of the EBOV genome ( Panel A ). During in-field sequencing we used a 19 reaction scheme or 11 reaction scheme which generated longer products. The predicted amplicon products are shown with forward primers and reverse primers indicated by green bars on the forward and reverse strand respectively, scaled according to the EBOV virus coordinates. The amplicon product sizes expected are shown for the 19 reaction scheme ( Panel B ) and the 11 reaction scheme ( Panel C ). No amplicon covers the extreme 3′ region of the genome. The last primer pair, 38_R, ends at position 18578, 381 bases away from the end of the virus genome. The primer diagram was created with Biopython 33 .
    Figure Legend Snippet: Primer schemes employed during the study We designed PCR primers to generate amplicons that would span the EBOV genome. We initially designed 38 primer pairs which were used in the initial validation study and which cover > 98% of the EBOV genome ( Panel A ). During in-field sequencing we used a 19 reaction scheme or 11 reaction scheme which generated longer products. The predicted amplicon products are shown with forward primers and reverse primers indicated by green bars on the forward and reverse strand respectively, scaled according to the EBOV virus coordinates. The amplicon product sizes expected are shown for the 19 reaction scheme ( Panel B ) and the 11 reaction scheme ( Panel C ). No amplicon covers the extreme 3′ region of the genome. The last primer pair, 38_R, ends at position 18578, 381 bases away from the end of the virus genome. The primer diagram was created with Biopython 33 .

    Techniques Used: Polymerase Chain Reaction, Sequencing, Generated, Amplification

    33) Product Images from "A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing"

    Article Title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep37563

    Process for efficient, zero-background, cloning of uniquely barcoded dA-tailed fragment libraries. (a) To achieve ligation of one unique fragment into each plasmid backbone, a relatively small cloning vector (5.7 kb) was generated containing a ccdB toxin gene flanked by two specifically tailored XcmI restriction enzyme cleavage sites. (b) The sequence is tailored so that XcmI enzyme digestion leaves a single 5′ dT-overhang on both open ends of the backbone generating a T-vector where the dA-tailed DNA fragments can be efficiently ligated without directional bias. (c) The cloning vector can have a flexible design with any 5′ and 3′ domain modulated by the inserted fragment. To allow for unique barcoding of each fragment together with the surrounding 5′ and 3′ domains, the region of interest in the cloning vector is then exposed to a two-step PCR amplification where a 5′ AttB1 site is inserted in the first amplification (20 cycles amplification). (d) The barcode together with the AttB2 site are added through a PCR with only a single elongation step, ensuring that each unique barcode is utilized only once and is not transferred to other amplicons due to PCR template switching. (e) The library of uniquely barcoded PCR amplicons is then inserted into the viral vector backbone using the “Gateway” BP clonase recombination reaction. (f) Chemically or electro-competent bacteria are then transformed and a small fraction of the transformation reaction plated for a rough estimation of total number of colonies. Generation of empty backbones ( i.e., missing a genomic fragment) is kept to an absolute minimal as any such plasmid would contain the ccdB toxin gene providing negative selection pressure.
    Figure Legend Snippet: Process for efficient, zero-background, cloning of uniquely barcoded dA-tailed fragment libraries. (a) To achieve ligation of one unique fragment into each plasmid backbone, a relatively small cloning vector (5.7 kb) was generated containing a ccdB toxin gene flanked by two specifically tailored XcmI restriction enzyme cleavage sites. (b) The sequence is tailored so that XcmI enzyme digestion leaves a single 5′ dT-overhang on both open ends of the backbone generating a T-vector where the dA-tailed DNA fragments can be efficiently ligated without directional bias. (c) The cloning vector can have a flexible design with any 5′ and 3′ domain modulated by the inserted fragment. To allow for unique barcoding of each fragment together with the surrounding 5′ and 3′ domains, the region of interest in the cloning vector is then exposed to a two-step PCR amplification where a 5′ AttB1 site is inserted in the first amplification (20 cycles amplification). (d) The barcode together with the AttB2 site are added through a PCR with only a single elongation step, ensuring that each unique barcode is utilized only once and is not transferred to other amplicons due to PCR template switching. (e) The library of uniquely barcoded PCR amplicons is then inserted into the viral vector backbone using the “Gateway” BP clonase recombination reaction. (f) Chemically or electro-competent bacteria are then transformed and a small fraction of the transformation reaction plated for a rough estimation of total number of colonies. Generation of empty backbones ( i.e., missing a genomic fragment) is kept to an absolute minimal as any such plasmid would contain the ccdB toxin gene providing negative selection pressure.

    Techniques Used: Clone Assay, Ligation, Plasmid Preparation, Generated, Sequencing, Polymerase Chain Reaction, Amplification, Transformation Assay, Selection

    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
    Figure Legend Snippet: Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.

    Techniques Used: Sequencing, Ligation, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Mutagenesis, Binding Assay, Generated

    Fragmentation of genetic sequences for insertion in barcode labelled plasmids. (a) A novel version of the Pfu-based proof-reading polymerase “Phusion U” allows for insertion of dUTP during PCR amplification. However, efficiency of amplification is reduced with increasing dUTP/dTTP fraction as exemplified through amplification of a genomic sequence (1.1 kb long) utilized in library 1. (b) The percentage dUTP used in the PCR reaction is determining the distribution of fragment sizes after nicking by Uracil-DNA-Glycosylase (UDG) and NaOH single strand breakage. (c) As the cleavage sites are strictly sequence dependent and statistically predictable based on dUTP fraction and insertion frequency it can be simulated using the NExTProg 1.0 software. Here this was modelled using the sequence for library 1. (d) The dUTP/UDG fragmentation protocol was applied to three amplicons with different sequences but similar length and GC content (49–52%), aimed for inclusion in library 3, and one shorter amplicon, with 10% dUTP. (e,f) The fragments from the first lane were further analysed by higher accuracy electrophoretic analysis (Bioanalyzer) and (g) Illumina sequencing was performed after insertion into a plasmid backbone (library 3).
    Figure Legend Snippet: Fragmentation of genetic sequences for insertion in barcode labelled plasmids. (a) A novel version of the Pfu-based proof-reading polymerase “Phusion U” allows for insertion of dUTP during PCR amplification. However, efficiency of amplification is reduced with increasing dUTP/dTTP fraction as exemplified through amplification of a genomic sequence (1.1 kb long) utilized in library 1. (b) The percentage dUTP used in the PCR reaction is determining the distribution of fragment sizes after nicking by Uracil-DNA-Glycosylase (UDG) and NaOH single strand breakage. (c) As the cleavage sites are strictly sequence dependent and statistically predictable based on dUTP fraction and insertion frequency it can be simulated using the NExTProg 1.0 software. Here this was modelled using the sequence for library 1. (d) The dUTP/UDG fragmentation protocol was applied to three amplicons with different sequences but similar length and GC content (49–52%), aimed for inclusion in library 3, and one shorter amplicon, with 10% dUTP. (e,f) The fragments from the first lane were further analysed by higher accuracy electrophoretic analysis (Bioanalyzer) and (g) Illumina sequencing was performed after insertion into a plasmid backbone (library 3).

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Software, Plasmid Preparation

    34) Product Images from "Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression"

    Article Title: Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to modulate chromatin structure and gene expression

    Journal: bioRxiv

    doi: 10.1101/484956

    Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).
    Figure Legend Snippet: Homologous pairing between repetitive DNA segments at the DSP site: a model. Topo II action at these sites leads to different consequences depending on the repeat orientations, direct and inverted. Plus-strand DNA path from 5’ to 3’ direction is depicted by black arrows. Homologous segments are shown by red arrows (upstream) and blue arrows (downstream) on plus strand or by dark-green arrows on minus strand. Homologous pairing between duplex DNAs aligned in parallel starts by ‘paranemic’ mode and converted to intertwined mode after topo II action. Left-handed crossovers facilitate the interaction between major grooves to form ‘recognition unit’, which is a quadruplex structure required for stable pairing [ 30 ]. Since the pairing occurs only when the two DNA segments are aligned in parallel, direct and inverted repeats bring about very different results both in the loop configuration between paired repeats and in the topological structure generated after topo II action. Direct and inverted repeats result in knotted loops and negative supercoils, respectively. The ‘crossover conversion’, which is an energetically favored step, is a mandatory process for the reverse reaction (unknotting or relaxation) to occur. The eTIP-seq experiments performed in the present study produced dominant DSP chimeras with RF/FR read orientations that are originated from direct repeats. This suggests that in terminally differentiating CGN cells topo IIβ is almost exclusively involved in unknotting reactions (illustrated in the upper right).

    Techniques Used: Generated, Produced

    Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.
    Figure Legend Snippet: Morphological observations implicating the involvement of topo IIβ in the differentiation of CGN in vitro . a Changes of chromatin structure. Nuclear DNA in fixed cells was stained with Hoechst 33342 at the culture days indicated. Nuclear images (z-stack with maximal diameter) were collected and arranged in 3 panels. b Similarity analysis by Wndchrm. Eighty images each were used for learning. Similarity indexes (between 0 and 1) shown are average of 4 trials. c Measurement of nuclear volume. Horizontal bars in boxplot indicate median values and p-values are from Mann-Whitney U test. d Degree of chromatin condensation as estimated from DNA staining intensities. The isosbestic point marked by arrow indicates the border between condensed and dispersed chromatin regions. Percentages of condensed region (right side) were box-plotted (inset). e Visualization of topo IIβ-SP120 interacting loci by proximity ligation assay (PLA) using culture day 2 (D2) cells. Z-projection images are shown. Scale bar, 1 µm. f Number of PLA foci plotted against the brightness of DNA stain where PLA resides. Total number of PLA signals used was 225. Dotted line marked by arrow indicates the borderline between condensed and dispersed chromatin regions. g Simultaneous detection of PLA and FISH signals. CGN cells at D5 were used and Z-projection images are shown. Arrows indicate the signals that are in contact or colocalized. Scale bar, 1 µm. h Close positioning of PLA and FISH spots during CGN differentiation (arrowheads). Scale bar, 1 µm. i Numerical analysis of FISH/PLA colocalization. See Methods for details. P-values were obtained by chi-squared test with Holm’s correction for multiple comparison. j Numerical change of PLA foci during differentiation. k Numerical change of FISH foci (Sat I) during differentiation. l Volumetric change of FISH foci (Sat I) during differentiation.

    Techniques Used: In Vitro, Staining, MANN-WHITNEY, Proximity Ligation Assay, Fluorescence In Situ Hybridization

    Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.
    Figure Legend Snippet: Overview of the eTIP-seq, a mapping technique used in the study. The topo IIβ-DNA complex illustrated in the box was isolated by eTIP procedure as described previously [ 11 ]. DNA in the topo IIβ-DNA complex captured on magnetic beads was then processed in either ways: fractionation by 0.5 M NaCl treatment (eTIPa-seq) or adaptor-mediated ligation (eTIPb-seq). Experimental results, data processing and toposite assignments for eTIPa-seq are summarized in Fig. S1a-d. Theoretical reasoning for the generation of three categories of toposites is shown in Fig. S2 by presenting all possible combinations of DNA fragments bound to topo IIβ. The eTIPb-seq procedure is outlined in Fig. S4a. See ‘Supplementary’ for detailed explanation on multiple forms of topo IIβ-DNA complex depicted in the box.

    Techniques Used: Isolation, Magnetic Beads, Fractionation, Ligation

    35) Product Images from "Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis"

    Article Title: Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/ncomms10063

    Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.
    Figure Legend Snippet: Timelines for sequencing-based analysis and culture-based DST. The timelines are shown for ( a ) S . aureus and ( b ) M . tuberculosis . In ( a ) both culture-based ( a ,i) and sequencing-based ( a ,ii) options involve 12 h of blood culture. After this, the culture-based approach (at Oxford University Hospitals clinical laboratory) follows with a direct coagulase test (Coag.) that provides a presumptive species identification at 4 h (marked ‘A'). Concurrently, blood culture is subcultured to blood agar, and MALDI-TOF confirms the species at 12 h (‘B'). A disc diffusion test for five antimicrobials (including methicillin) is performed directly from a positive blood culture providing first-line susceptibility information 18–24 h later (‘C'), assuming an acceptable inoculum. Finally, post-subculture samples are undergo extended susceptibility testing by automated broth microdilution (brandname ‘Phoenix'), giving final results after another 18–24 h (‘D'). For the sequencing-based workflow ( a ,ii), the DNA extraction plus sample preparation takes 7.5 h because samples are from blood culture, not colony isolates. With the Illumina MiSeq v3 reagents, a 16.5 h run is possible (giving paired 75 bp reads, adequate for this purpose), giving full susceptibility results at the same time as direct disc tests provide results for five drugs. ( b ) The culture-based process ( b ,i; in a typical UK reference laboratory) starts with two weeks of mycobacterial growth indicator tube (MGIT) culture, followed by a species identification test (‘X'). If the species belongs to the MTBC, then DST is run in MGIT, and at decision point ‘Y', if the sample tests susceptible to all first-line drugs, no further testing is done. MGIT DST is repeated for pyrazinamide if the first test revealed resistance to this drug. If there is resistance to any other drug, then solid culture DST is performed. If these tests show there is resistance to rifampicin then another round of MGIT culture followed by MGIT DST is done for second-line drugs. For sequencing-based approaches we show timelines for the present study ( b ,ii) and a potential alternative ( b ,iii), which would reduce time-to-results to just over 2 weeks.

    Techniques Used: Sequencing, Diffusion-based Assay, DNA Extraction, Sample Prep

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