Structured Review

Illumina Inc amplicons
(a) PCR <t>amplicons</t> of the T7 promoter oligo pool can be seen as the strong bands around 130 bp (4% Agarose E-Gel EX with Low Range Quantitative DNA Ladder). (b) Serial dilutions of the processed single stranded T7 promoter oligo library (left 4 lanes) compared to a reference oligo of 90 bp (right 4 lanes), which indicates correct processing of the oligos from 130 bp to 90 bp oligos ready for MAGE (TBE-Urea gel 4% from Invitrogen). (c) Comparison of MAGE efficiency using column-synthesized oligos and microarray-processed oligos by MO-MAGE. Gel shows size distribution of the two processed oligo pools (TBE-Urea gel 4% from Invitrogen).
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Images

1) Product Images from "Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides"

Article Title: Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides

Journal: ACS Synthetic Biology

doi: 10.1021/sb5001565

(a) PCR amplicons of the T7 promoter oligo pool can be seen as the strong bands around 130 bp (4% Agarose E-Gel EX with Low Range Quantitative DNA Ladder). (b) Serial dilutions of the processed single stranded T7 promoter oligo library (left 4 lanes) compared to a reference oligo of 90 bp (right 4 lanes), which indicates correct processing of the oligos from 130 bp to 90 bp oligos ready for MAGE (TBE-Urea gel 4% from Invitrogen). (c) Comparison of MAGE efficiency using column-synthesized oligos and microarray-processed oligos by MO-MAGE. Gel shows size distribution of the two processed oligo pools (TBE-Urea gel 4% from Invitrogen).
Figure Legend Snippet: (a) PCR amplicons of the T7 promoter oligo pool can be seen as the strong bands around 130 bp (4% Agarose E-Gel EX with Low Range Quantitative DNA Ladder). (b) Serial dilutions of the processed single stranded T7 promoter oligo library (left 4 lanes) compared to a reference oligo of 90 bp (right 4 lanes), which indicates correct processing of the oligos from 130 bp to 90 bp oligos ready for MAGE (TBE-Urea gel 4% from Invitrogen). (c) Comparison of MAGE efficiency using column-synthesized oligos and microarray-processed oligos by MO-MAGE. Gel shows size distribution of the two processed oligo pools (TBE-Urea gel 4% from Invitrogen).

Techniques Used: Polymerase Chain Reaction, Synthesized, Microarray

2) Product Images from "Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario"

Article Title: Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario

Journal: Virus research

doi: 10.1016/j.virusres.2017.02.007

(a) A schematic of the RRV genome indicating the position of the five ORFs above which the locations of the 1 st and 2 nd round amplicons are shown. The PCR primers used are indicated at the ends of each amplicon while the size of each product is given
Figure Legend Snippet: (a) A schematic of the RRV genome indicating the position of the five ORFs above which the locations of the 1 st and 2 nd round amplicons are shown. The PCR primers used are indicated at the ends of each amplicon while the size of each product is given

Techniques Used: Polymerase Chain Reaction, Amplification

3) Product Images from "Fragmented mitochondrial genomes of the rat lice, Polyplax asiatica and Polyplax spinulosa: intra-genus variation in fragmentation pattern and a possible link between the extent of fragmentation and the length of life cycle"

Article Title: Fragmented mitochondrial genomes of the rat lice, Polyplax asiatica and Polyplax spinulosa: intra-genus variation in fragmentation pattern and a possible link between the extent of fragmentation and the length of life cycle

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-44

PCR amplification of the mitochondrial (mt) minichromosomes of the Polyplax rat lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 2: PCR amplicons generated with primer pair 57F-57R that spans the coding region of each mitochondrial (mt) minichromosome of Polyplax asiatica . Lane 4: PCR amplicons generated with primer pair 301F-301R that spans the coding region of each mt minichromosome of Polyplax spinulosa . (B) PCR verification of the mt minichromosomes of Po. asiatica . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. asiatica : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 2 ( taa ), trnD - trnY - cox2 - nad6 , trnR - nad4L - cox3 - trnA , trnS 1 ( tct )- trnS 2 ( tga )- nad1 - trnT - trnG - nad3 - trnW , trnQ - nad2 - trnN - trnP , trnK - nad4 - trnF , trnH - nad5 , rrnS - trnC , trnM - trnL 1 ( tag )- rrnL - trnV . (C) PCR verification of the mt minichromosomes of Po. spinulosa . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. spinulosa : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 1 ( tag ), trnT - trnD - trnY - cox2 - nad6 - trnA , trnR - nad4L - trnP - cox3 , nad1 - trnG - nad3 - trnW , trnQ - nad2 - trnN , trnK - nad4 , trnH - nad5 - trnF , trnS 1 ( tct )- trnS 2 ( tga )- rrnS - trnC , trnM - trnL 2 ( taa )- rrnL - trnV . Genes from which PCR primers were designed are in bold.
Figure Legend Snippet: PCR amplification of the mitochondrial (mt) minichromosomes of the Polyplax rat lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 2: PCR amplicons generated with primer pair 57F-57R that spans the coding region of each mitochondrial (mt) minichromosome of Polyplax asiatica . Lane 4: PCR amplicons generated with primer pair 301F-301R that spans the coding region of each mt minichromosome of Polyplax spinulosa . (B) PCR verification of the mt minichromosomes of Po. asiatica . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. asiatica : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 2 ( taa ), trnD - trnY - cox2 - nad6 , trnR - nad4L - cox3 - trnA , trnS 1 ( tct )- trnS 2 ( tga )- nad1 - trnT - trnG - nad3 - trnW , trnQ - nad2 - trnN - trnP , trnK - nad4 - trnF , trnH - nad5 , rrnS - trnC , trnM - trnL 1 ( tag )- rrnL - trnV . (C) PCR verification of the mt minichromosomes of Po. spinulosa . Lane 1 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2–12: PCR amplicons from the 11 minichromosomes of Po. spinulosa : atp8 - atp6 , trnE - cob - trnI , cox1 - trnL 1 ( tag ), trnT - trnD - trnY - cox2 - nad6 - trnA , trnR - nad4L - trnP - cox3 , nad1 - trnG - nad3 - trnW , trnQ - nad2 - trnN , trnK - nad4 , trnH - nad5 - trnF , trnS 1 ( tct )- trnS 2 ( tga )- rrnS - trnC , trnM - trnL 2 ( taa )- rrnL - trnV . Genes from which PCR primers were designed are in bold.

Techniques Used: Polymerase Chain Reaction, Amplification, Generated

4) Product Images from "A method for high‐throughput production of sequence‐verified DNA libraries and strain collections"

Article Title: A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

Journal: Molecular Systems Biology

doi: 10.15252/msb.20167233

Recombinase Directed Indexing (REDI) for high‐quality DNA libraries (1) A complex library (e.g. array‐synthesized oligonucleotide DNA) is amplified by PCR and integrated into the yeast genome by transformation and homologous recombination. (2) MAT α transformants are arrayed in 1,536 format and (3) mated to 1,536 unique MAT a barcoder strains. (4) Site‐specific (Cre‐lox) recombination in diploids physically links the barcode to the exogenous oligonucleotide DNA. (5) Diploid recombinants are combined, and the barcode oligonucleotide locus is amplified by PCR using common priming sites. (6) Amplicons are subjected to paired‐end Illumina sequencing. As the barcode assigned to each MAT a barcoder strain is known, the plate position of MAT α transformants containing oligonucleotide DNA of interest can be readily identified from the sequencing results. (7) Clones are selected and applied in downstream applications of interest. The approximate time required for each step (including yeast growth time) is indicated in parentheses, and is based on processing ~10,000 transformants and isolating ~5,000 clones of interest. Steps (2) and (7) would require additional time if more clones are processed. * indicates DNA synthesis errors.
Figure Legend Snippet: Recombinase Directed Indexing (REDI) for high‐quality DNA libraries (1) A complex library (e.g. array‐synthesized oligonucleotide DNA) is amplified by PCR and integrated into the yeast genome by transformation and homologous recombination. (2) MAT α transformants are arrayed in 1,536 format and (3) mated to 1,536 unique MAT a barcoder strains. (4) Site‐specific (Cre‐lox) recombination in diploids physically links the barcode to the exogenous oligonucleotide DNA. (5) Diploid recombinants are combined, and the barcode oligonucleotide locus is amplified by PCR using common priming sites. (6) Amplicons are subjected to paired‐end Illumina sequencing. As the barcode assigned to each MAT a barcoder strain is known, the plate position of MAT α transformants containing oligonucleotide DNA of interest can be readily identified from the sequencing results. (7) Clones are selected and applied in downstream applications of interest. The approximate time required for each step (including yeast growth time) is indicated in parentheses, and is based on processing ~10,000 transformants and isolating ~5,000 clones of interest. Steps (2) and (7) would require additional time if more clones are processed. * indicates DNA synthesis errors.

Techniques Used: Synthesized, Amplification, Polymerase Chain Reaction, Transformation Assay, Homologous Recombination, Sequencing, Clone Assay, DNA Synthesis

5) Product Images from "2.7 million samples genotyped for HLA by next generation sequencing: lessons learned"

Article Title: 2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

Journal: BMC Genomics

doi: 10.1186/s12864-017-3575-z

Proportion of PCR-mediated recombinant reads (chimeric reads) for different HLA amplicons and different workflows (see Methods for details)
Figure Legend Snippet: Proportion of PCR-mediated recombinant reads (chimeric reads) for different HLA amplicons and different workflows (see Methods for details)

Techniques Used: Polymerase Chain Reaction, Recombinant

Distribution of on-target paired-end reads across amplicons. Different colours indicate different HLA loci. Solid lines indicate exon 2 amplicons; dashed lines indicate exon 3 amplicons
Figure Legend Snippet: Distribution of on-target paired-end reads across amplicons. Different colours indicate different HLA loci. Solid lines indicate exon 2 amplicons; dashed lines indicate exon 3 amplicons

Techniques Used:

6) Product Images from "Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing"

Article Title: Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.01294

Microbiota and major flavor components analysis across all the samples. Average bacterial (A) and fungal (B) distribution at the genus-level in microbiota based on 16S rRNA gene and ITS amplicons ( n = 18, each bar n = 3). (C) Amplicons analysis represented the similarities of microbial compositions based on principal component analysis (PCA). (D) Ethanol, lactic acid, and acetic acid production had different stages. Bars represented mean (±SE). Asterisk indicates significant differences by Mann–Whitney U test ( P
Figure Legend Snippet: Microbiota and major flavor components analysis across all the samples. Average bacterial (A) and fungal (B) distribution at the genus-level in microbiota based on 16S rRNA gene and ITS amplicons ( n = 18, each bar n = 3). (C) Amplicons analysis represented the similarities of microbial compositions based on principal component analysis (PCA). (D) Ethanol, lactic acid, and acetic acid production had different stages. Bars represented mean (±SE). Asterisk indicates significant differences by Mann–Whitney U test ( P

Techniques Used: MANN-WHITNEY

7) Product Images from "Anaerobic digestion of pig manure supernatant at high ammonia concentrations characterized by high abundances of Methanosaeta and non-euryarchaeotal archaea"

Article Title: Anaerobic digestion of pig manure supernatant at high ammonia concentrations characterized by high abundances of Methanosaeta and non-euryarchaeotal archaea

Journal: Scientific Reports

doi: 10.1038/s41598-017-14527-1

Relative abundances of bacterial classes ( A ) and archaeal genera ( B ) represented in the v3–4 16S rDNA amplicons obtained for individual reactor samples. Each bar represents one sample, and is labelled as follows: D indicates the experimental day; HA1 and HA2 refer to the two reactors operated at 3.7 ± 0.2 g NH 4 -N L −1 , and LA1, and LA2 refers to the reactors operated at 1.9 ± 0.2 g NH 4 -N L −1 . OTUs that could not be classified at the domain level are labeled “Unclassified”, while OTUs that could not be classified at class or genus level for bacteria and archaea, respectively, are labeled *. Only taxa represented by a portion of ≥1% of the sequence reads in at least one of the samples are shown. “Others” include all reads representing the taxa with
Figure Legend Snippet: Relative abundances of bacterial classes ( A ) and archaeal genera ( B ) represented in the v3–4 16S rDNA amplicons obtained for individual reactor samples. Each bar represents one sample, and is labelled as follows: D indicates the experimental day; HA1 and HA2 refer to the two reactors operated at 3.7 ± 0.2 g NH 4 -N L −1 , and LA1, and LA2 refers to the reactors operated at 1.9 ± 0.2 g NH 4 -N L −1 . OTUs that could not be classified at the domain level are labeled “Unclassified”, while OTUs that could not be classified at class or genus level for bacteria and archaea, respectively, are labeled *. Only taxa represented by a portion of ≥1% of the sequence reads in at least one of the samples are shown. “Others” include all reads representing the taxa with

Techniques Used: Labeling, Sequencing

8) Product Images from "Microbial Community Composition and Functional Capacity in a Terrestrial Ferruginous, Sulfate-Depleted Mud Volcano"

Article Title: Microbial Community Composition and Functional Capacity in a Terrestrial Ferruginous, Sulfate-Depleted Mud Volcano

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02137

Community compositions and variations revealed by 16S rRNA gene amplicons. (A) Non-metric multidimensional scaling of community relatedness quantified by the weighted Unifrac matrix with the overlay of ordination for selected environmental parameters. (B) Abundances of major divisions for environmental and incubated samples.
Figure Legend Snippet: Community compositions and variations revealed by 16S rRNA gene amplicons. (A) Non-metric multidimensional scaling of community relatedness quantified by the weighted Unifrac matrix with the overlay of ordination for selected environmental parameters. (B) Abundances of major divisions for environmental and incubated samples.

Techniques Used: Incubation

9) Product Images from "Increased Biosynthetic Gene Dosage in a Genome-Reduced Defensive Bacterial Symbiont"

Article Title: Increased Biosynthetic Gene Dosage in a Genome-Reduced Defensive Bacterial Symbiont

Journal: mSystems

doi: 10.1128/mSystems.00096-17

(A) Scale map of Lissoclinum sp. mitochondrion genome and comparison to the genome of the Lissoclinum patella L2 mitochondrion. (B and C) Approximately maximum-likelihood trees based on two nonoverlapping amplicons in the mitochondrial cytochrome c oxidase I (COX1) gene. Bootstrap proportions greater than 70% are expressed to the left of each node as a percentage of 1,000 replicates. Sequence identities of the Lissoclinum sp. COX1 gene to its close relatives are shown in Fig. S8 .
Figure Legend Snippet: (A) Scale map of Lissoclinum sp. mitochondrion genome and comparison to the genome of the Lissoclinum patella L2 mitochondrion. (B and C) Approximately maximum-likelihood trees based on two nonoverlapping amplicons in the mitochondrial cytochrome c oxidase I (COX1) gene. Bootstrap proportions greater than 70% are expressed to the left of each node as a percentage of 1,000 replicates. Sequence identities of the Lissoclinum sp. COX1 gene to its close relatives are shown in Fig. S8 .

Techniques Used: Sequencing

10) Product Images from "A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function"

Article Title: A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function

Journal: Scientific Reports

doi: 10.1038/s41598-018-24036-4

duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa Δ11 carriers. Full-length gel is shown in Supplementary Fig. 7 from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.
Figure Legend Snippet: duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa Δ11 carriers. Full-length gel is shown in Supplementary Fig. 7 from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.

Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Polyacrylamide Gel Electrophoresis, Immunofluorescence, Staining

11) Product Images from "Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes"

Article Title: Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2017.03.021

Sequence confirmation of DDC-mRuby knock-in allele. (A) Schematic showing the DDC-VA-P2A-mRuby-3X NLS knock-in allele and the location of genotyping PCR primers. Two PCR reactions, one each for 5′ and 3′ junction of the insert, were used for assessing the insertions. The PCR amplicon sizes are shown above the schematic and the primer sequences below the schematic. (B) Genotyping agarose gel images showing correct insertions at 5′ (top panel) and 3′ (bottom panel). Animals #8, #10 and #11 show expected amplicons for both the junction PCRs. Sequencing of the PCR products showed that animals #8 and #10 had indels or mutations whereas the animal #11 had precise insertions at both the junctions without any mutations. (C) Sequencing of the insertion junctions of the animal #11 showing precise insertion of the cassette at the desired site (immediately before the stop codon); compare with the sequences shown in Fig. 6C and D .
Figure Legend Snippet: Sequence confirmation of DDC-mRuby knock-in allele. (A) Schematic showing the DDC-VA-P2A-mRuby-3X NLS knock-in allele and the location of genotyping PCR primers. Two PCR reactions, one each for 5′ and 3′ junction of the insert, were used for assessing the insertions. The PCR amplicon sizes are shown above the schematic and the primer sequences below the schematic. (B) Genotyping agarose gel images showing correct insertions at 5′ (top panel) and 3′ (bottom panel). Animals #8, #10 and #11 show expected amplicons for both the junction PCRs. Sequencing of the PCR products showed that animals #8 and #10 had indels or mutations whereas the animal #11 had precise insertions at both the junctions without any mutations. (C) Sequencing of the insertion junctions of the animal #11 showing precise insertion of the cassette at the desired site (immediately before the stop codon); compare with the sequences shown in Fig. 6C and D .

Techniques Used: Sequencing, Knock-In, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

12) Product Images from "Complementary Amplicon-Based Genomic Approaches for the Study of Fungal Communities in Humans"

Article Title: Complementary Amplicon-Based Genomic Approaches for the Study of Fungal Communities in Humans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0116705

Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .
Figure Legend Snippet: Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing

13) Product Images from "Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations"

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

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

doi: 10.1073/pnas.1701382114

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

Techniques Used: Amplification

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

Techniques Used: Amplification

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

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

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

Techniques Used: Amplification

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

Techniques Used:

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

Techniques Used: Amplification

14) Product Images from "Evolutionary analysis of the Chikungunya virus epidemic in Mexico reveals intra-host mutational hotspots in the E1 protein"

Article Title: Evolutionary analysis of the Chikungunya virus epidemic in Mexico reveals intra-host mutational hotspots in the E1 protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209292

Abundance of viral haplotypes in two E1 gene amplicons. Heat maps indicating the relative abundance (red colour intensity) of distinct amino acid viral haplotypes following deep sequencing of two amplicons COF 1 (A) and CIF 2 (B) that span the near full-length CHIKV E1 gene in 25 Mexican patients. Dendrograms represent clustering of patients according to haplotype diversity.
Figure Legend Snippet: Abundance of viral haplotypes in two E1 gene amplicons. Heat maps indicating the relative abundance (red colour intensity) of distinct amino acid viral haplotypes following deep sequencing of two amplicons COF 1 (A) and CIF 2 (B) that span the near full-length CHIKV E1 gene in 25 Mexican patients. Dendrograms represent clustering of patients according to haplotype diversity.

Techniques Used: Sequencing

15) Product Images from "Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing"

Article Title: Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing

Journal: Journal of Virology

doi: 10.1128/JVI.03391-14

(A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.
Figure Legend Snippet: (A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.

Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Sequencing

16) Product Images from "Fragmented mitochondrial genomes are present in both major clades of the blood-sucking lice (suborder Anoplura): evidence from two Hoplopleura rodent lice (family Hoplopleuridae)"

Article Title: Fragmented mitochondrial genomes are present in both major clades of the blood-sucking lice (suborder Anoplura): evidence from two Hoplopleura rodent lice (family Hoplopleuridae)

Journal: BMC Genomics

doi: 10.1186/1471-2164-15-751

PCR amplification of the mitochondrial (mt) minichromosomes of the Hoplopleura rodent lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 3: PCR amplicons generated with primer pair 249F-249R that spans the coding region of each mt minichromosome of Hoplopleura akanezumi . Lane 5: PCR amplicons generated with primer pair 344F-344R that spans the coding region of each mt minichromosome of Hoplopleura kitti . (B) PCR verification of the mt minichromosomes of Ho. akanezumi . Lane 1, 10 and 14: 500 bp DNA Ladder (TIANGEN). Lane 2-13: PCR amplicons from the 10 minichromosomes and a chimeric mt minichromosome of Ho. akanezumi : atp8-atp6- trnN , trnE-cob- trnS 1 -trnS 2 , trnI- cox1 , trnD-trnY 1 - cox2 , trnR-nad4L-trnP- cox3 -trnA-trnT , nad2 , trnK- nad4 , trnC- nad6 -trnW-trnL 2 (taa) , rrnS , trnY 2 - rrnL -trnV , chimeric trnY 2 - prrnL - prrnS . (C) PCR verification of the mt minichromosomes of Ho. kitti . Lane 1, 10 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2-13: PCR amplicons from the 11 minichromosomes of Ho. kitti : atp8-atp6- trnN , trnE -cob-trnS 1 -trnS 2 , trnI - cox1 , trnD-trnY- cox2 -trnT , trnR-nad4L-trnP- cox3 -trnA , trnQ- nad1 -trnG-nad3 , nad2 , trnK -nad4 , trnC- nad6 -trnW-trnL 2 (taa) , rrnS , trnM-trnL 1 (tag)- rrnL -trnV . Genes from which PCR primers were designed are in bold.
Figure Legend Snippet: PCR amplification of the mitochondrial (mt) minichromosomes of the Hoplopleura rodent lice. (A) Lane 1: GeneRuler®100 bp DNA Ladder (Thermo Scientific). Lane 3: PCR amplicons generated with primer pair 249F-249R that spans the coding region of each mt minichromosome of Hoplopleura akanezumi . Lane 5: PCR amplicons generated with primer pair 344F-344R that spans the coding region of each mt minichromosome of Hoplopleura kitti . (B) PCR verification of the mt minichromosomes of Ho. akanezumi . Lane 1, 10 and 14: 500 bp DNA Ladder (TIANGEN). Lane 2-13: PCR amplicons from the 10 minichromosomes and a chimeric mt minichromosome of Ho. akanezumi : atp8-atp6- trnN , trnE-cob- trnS 1 -trnS 2 , trnI- cox1 , trnD-trnY 1 - cox2 , trnR-nad4L-trnP- cox3 -trnA-trnT , nad2 , trnK- nad4 , trnC- nad6 -trnW-trnL 2 (taa) , rrnS , trnY 2 - rrnL -trnV , chimeric trnY 2 - prrnL - prrnS . (C) PCR verification of the mt minichromosomes of Ho. kitti . Lane 1, 10 and 13: 500 bp DNA Ladder (TIANGEN). Lane 2-13: PCR amplicons from the 11 minichromosomes of Ho. kitti : atp8-atp6- trnN , trnE -cob-trnS 1 -trnS 2 , trnI - cox1 , trnD-trnY- cox2 -trnT , trnR-nad4L-trnP- cox3 -trnA , trnQ- nad1 -trnG-nad3 , nad2 , trnK -nad4 , trnC- nad6 -trnW-trnL 2 (taa) , rrnS , trnM-trnL 1 (tag)- rrnL -trnV . Genes from which PCR primers were designed are in bold.

Techniques Used: Polymerase Chain Reaction, Amplification, Generated

17) Product Images from "Identification of novel HNF1B mRNA splicing variants and their qualitative and semi-quantitative profile in selected healthy and tumour tissues"

Article Title: Identification of novel HNF1B mRNA splicing variants and their qualitative and semi-quantitative profile in selected healthy and tumour tissues

Journal: Scientific Reports

doi: 10.1038/s41598-020-63733-x

Overview of the methodical approach. ( A ) Scheme of the sample pools processing. cDNA samples from 32 individuals were mixed by four into 11 pools according to tissue type (1 – endometrial endometrioid carcinoma, 2 – colorectal carcinoma, 3 – paired colorectal healthy tissue, 4 – kidney carcinoma, 5 – paired healthy kidney, 6 – kidney oncocytoma, 7 – pancreatic carcinoma, 8 – paired healthy pancreas, 9 – prostate carcinoma, 10 – paired healthy tissue (seminal vesicles), 11 – prostate hyperplasia. ( B ) Scheme of the mPCR primer locations across the full-length HNF1B transcript (Red = forward primers; blue = reverse primers). In exon 3, an additional reverse primer was designed to cover the known HNF1B 3p variant (Fig. 1 ). ( C ) Scheme of 7 mPCR reactions with respective primer usage. The used mPCR reactions are designed to cover the amplification of all possible exon-exon junctions. ( D ) An example of the electropherograms from capillary electrophoresis of the final mPCR mixture of the healthy prostate pool. The red dashed line box represents the area of our interest with short amplicons raised from alternative splicing, the violet dashed line box represents the area with amplicons raised from the canonical transcripts. ( E ) Capillary electrophoresis of the prepared sequencing library of all mPCR pools. Same description applies for the dashed line boxes as for Fig. 2D . Comparing the size of the peaks between ( D and E ) shows enrichment of short amplicons after size selection (red dashed line box), and sequencing adaptor ligation adds +166 bp in amplicon length. ( F ) Manual analysis of the mapped reads in IGV viewer, example of the variant HNF1BΔ7. The left (red) part corresponds to the sequence of exon 6 and the right (blue) part of the amplicons corresponds to the sequence of exon 8; the number of reads was deducted and scored (the yellow pop-up box) using the grey IGV coverage bar.
Figure Legend Snippet: Overview of the methodical approach. ( A ) Scheme of the sample pools processing. cDNA samples from 32 individuals were mixed by four into 11 pools according to tissue type (1 – endometrial endometrioid carcinoma, 2 – colorectal carcinoma, 3 – paired colorectal healthy tissue, 4 – kidney carcinoma, 5 – paired healthy kidney, 6 – kidney oncocytoma, 7 – pancreatic carcinoma, 8 – paired healthy pancreas, 9 – prostate carcinoma, 10 – paired healthy tissue (seminal vesicles), 11 – prostate hyperplasia. ( B ) Scheme of the mPCR primer locations across the full-length HNF1B transcript (Red = forward primers; blue = reverse primers). In exon 3, an additional reverse primer was designed to cover the known HNF1B 3p variant (Fig. 1 ). ( C ) Scheme of 7 mPCR reactions with respective primer usage. The used mPCR reactions are designed to cover the amplification of all possible exon-exon junctions. ( D ) An example of the electropherograms from capillary electrophoresis of the final mPCR mixture of the healthy prostate pool. The red dashed line box represents the area of our interest with short amplicons raised from alternative splicing, the violet dashed line box represents the area with amplicons raised from the canonical transcripts. ( E ) Capillary electrophoresis of the prepared sequencing library of all mPCR pools. Same description applies for the dashed line boxes as for Fig. 2D . Comparing the size of the peaks between ( D and E ) shows enrichment of short amplicons after size selection (red dashed line box), and sequencing adaptor ligation adds +166 bp in amplicon length. ( F ) Manual analysis of the mapped reads in IGV viewer, example of the variant HNF1BΔ7. The left (red) part corresponds to the sequence of exon 6 and the right (blue) part of the amplicons corresponds to the sequence of exon 8; the number of reads was deducted and scored (the yellow pop-up box) using the grey IGV coverage bar.

Techniques Used: Variant Assay, Amplification, Electrophoresis, Sequencing, Selection, Ligation

18) Product Images from "Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage"

Article Title: Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage

Journal: bioRxiv

doi: 10.1101/2020.05.05.078386

Chromosomal location of all amplicons and corresponding DNA methylation levels Chromosomal position of all genes are illustrated using graphical outputs generated by the UCSC Genome Browser ( https://genome.ucsc.edu ) and Geneious Prime 2019 software ( https://www.geneious.com ). All genes are presented in 5’ - > 3’ orientation from left to right. Amplicons are highlighted with respect to their genomic orientation. All CpG sites are illustrated with green bars, CpG islands in light green, and exons in grey color. To determine the reliability of the assay, genomic DNA of a male and female donor were both bisulfite-treated in triplicates. From each triplicate and a pool of the triplicates 10 PCR replicates were prepared from all amplicons to test for the means standard deviations of the assays. Linearity of the assays is illustrated by plotting expected to experimental methylation values of a serial diluted methylation standard.
Figure Legend Snippet: Chromosomal location of all amplicons and corresponding DNA methylation levels Chromosomal position of all genes are illustrated using graphical outputs generated by the UCSC Genome Browser ( https://genome.ucsc.edu ) and Geneious Prime 2019 software ( https://www.geneious.com ). All genes are presented in 5’ - > 3’ orientation from left to right. Amplicons are highlighted with respect to their genomic orientation. All CpG sites are illustrated with green bars, CpG islands in light green, and exons in grey color. To determine the reliability of the assay, genomic DNA of a male and female donor were both bisulfite-treated in triplicates. From each triplicate and a pool of the triplicates 10 PCR replicates were prepared from all amplicons to test for the means standard deviations of the assays. Linearity of the assays is illustrated by plotting expected to experimental methylation values of a serial diluted methylation standard.

Techniques Used: DNA Methylation Assay, Generated, Software, Polymerase Chain Reaction, Methylation

DNA methylation levels comparing BPD to healthy controls Percentage methylation and standard deviation of all CpGs are presented for all amplicons/genes comparing BPD patients (grey) vs healthy controls (black)
Figure Legend Snippet: DNA methylation levels comparing BPD to healthy controls Percentage methylation and standard deviation of all CpGs are presented for all amplicons/genes comparing BPD patients (grey) vs healthy controls (black)

Techniques Used: DNA Methylation Assay, Methylation, Standard Deviation

19) Product Images from "Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline"

Article Title: Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Journal: bioRxiv

doi: 10.1101/2020.03.11.986174

Agilent TapeStation 4200 results of RNA fragementation using High Sensitivity RNA ScreenTape. A small aliquot of total RNA (2μg) was fragemented for various durations at 70C and then a highly-diluted sample (~3ng/μL) was analyzed according to the manufacturer’s protocol. A) Example of over-fragmented sample that is outside the recommended range of 100-200bp (9 min of fragmentation). B) Optimal fragmentation result (~3 min). C) Under fragmented sample (1 min with higher concentration); although minimal under-fragmenation will likely still yield acceptable libraries, more extreme scenarios such as the one depicted will result in large amplicons that are outside the recommended range of the sequencer. “Lower” refers to the lower size marker for the TapeStation instrument.
Figure Legend Snippet: Agilent TapeStation 4200 results of RNA fragementation using High Sensitivity RNA ScreenTape. A small aliquot of total RNA (2μg) was fragemented for various durations at 70C and then a highly-diluted sample (~3ng/μL) was analyzed according to the manufacturer’s protocol. A) Example of over-fragmented sample that is outside the recommended range of 100-200bp (9 min of fragmentation). B) Optimal fragmentation result (~3 min). C) Under fragmented sample (1 min with higher concentration); although minimal under-fragmenation will likely still yield acceptable libraries, more extreme scenarios such as the one depicted will result in large amplicons that are outside the recommended range of the sequencer. “Lower” refers to the lower size marker for the TapeStation instrument.

Techniques Used: Concentration Assay, Marker

20) Product Images from "Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy"

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

Journal: Nature Communications

doi: 10.1038/ncomms14454

In vivo gene editing introduces a functional ORF in mdx 4cv mouse muscles. ( a ) Deep sequencing quantification on PCR amplicons generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n =4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. ( b ) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n =4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. ( c ) Subclone sequencing of the treatment-specific RT–PCR product (red box in b ) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). ( d ) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n =5) and RT–PCR amplicons generated from pooled transcripts (right, n =4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. ( e ) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).
Figure Legend Snippet: In vivo gene editing introduces a functional ORF in mdx 4cv mouse muscles. ( a ) Deep sequencing quantification on PCR amplicons generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n =4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. ( b ) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n =4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. ( c ) Subclone sequencing of the treatment-specific RT–PCR product (red box in b ) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). ( d ) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n =5) and RT–PCR amplicons generated from pooled transcripts (right, n =4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. ( e ) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).

Techniques Used: In Vivo, Functional Assay, Sequencing, Polymerase Chain Reaction, Generated, Non-Homologous End Joining, Reverse Transcription Polymerase Chain Reaction, Isolation

21) Product Images from "Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism"

Article Title: Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2019.00273

Insert-trap PCR detects MT-int insertion events at the Ctr9 CRISPR site. (A) Schematic showing the insert-trap PCR strategy for finding the 3′-end of an MT-int insert expected at the Ctr9 CRISPR locus. There are two possible orientations of MT-int DNA insertion, forward and reverse; the use of the red (insert primer) and green (R1) primer set detects the forward MT-int insert only. (B) High frequency of MT-int DNA insertion at the Ctr9 CRISPR site. PCR was performed using as templates the primary PCR products (see Figure 1A ) that failed to produce relevant amplicons in the subsequent nested PCR. PCR products of different sizes generated by insert-trap PCR are shown along with the reference amplicon of the sample in Figure 4A (left). The identities and structures of the inserts are schematically represented after sequencing (right). (C) Similarity in DNA sequence at the insertion sites. The sequence 5′-AGACCA-3′ appears frequently at the insertion sites. The green brackets and dots indicate deletion regions.
Figure Legend Snippet: Insert-trap PCR detects MT-int insertion events at the Ctr9 CRISPR site. (A) Schematic showing the insert-trap PCR strategy for finding the 3′-end of an MT-int insert expected at the Ctr9 CRISPR locus. There are two possible orientations of MT-int DNA insertion, forward and reverse; the use of the red (insert primer) and green (R1) primer set detects the forward MT-int insert only. (B) High frequency of MT-int DNA insertion at the Ctr9 CRISPR site. PCR was performed using as templates the primary PCR products (see Figure 1A ) that failed to produce relevant amplicons in the subsequent nested PCR. PCR products of different sizes generated by insert-trap PCR are shown along with the reference amplicon of the sample in Figure 4A (left). The identities and structures of the inserts are schematically represented after sequencing (right). (C) Similarity in DNA sequence at the insertion sites. The sequence 5′-AGACCA-3′ appears frequently at the insertion sites. The green brackets and dots indicate deletion regions.

Techniques Used: Polymerase Chain Reaction, CRISPR, Nested PCR, Generated, Amplification, Sequencing

Indel patterns and frequencies in mouse 8-cell embryos and embryonic stem cells derived from CRISP/Cas9-mediated editing of mouse zygotes. (A) Strategy for PCR genotyping. Eight-cell embryos derived from the microinjection of sgRNAs and recombinant Cas9 proteins into the pronuclei of mouse zygotes were disassembled to obtain single blastomeres before primary PCR to simultaneously detect the Gapdh and either the Ctr9 or the Brca2 loci. Nested or hemi-nested PCR (2° PCR) was performed using the primary PCR product, and the resulting product was combined and resolved on a polyacrylamide gel (PAGE). (B) Determination of the Ctr9 (left) and Brca2 (right) genotypes in individual blastomeres of 8-cell embryos (A to I, n = 9) based on the sizes of PCR products. Each PAGE represents a single embryo containing eight blastomeres (numbered 1-8); individual lanes are marked by “v” and “a” based on the types of alleles they contain (indel and normal, respectively). Lanes devoid of Ctr9 or Brca2 PCR product are either marked by “x” or unmarked depending on the presence or absence of the Gapdh PCR band, respectively. The blastomeres in the Ctr9 “B” embryo exhibited more PCR bands than would be expected from a diploid cell, hinting at a polyploid (Angell et al., 1987 ; Munne et al., 1994 ). The additional PCR bands at higher positions (for instance, those in the B3, B6, D5, F3, F4, H1, H3, and I6 Ctr9 embryos), especially in the blastomeres carrying heterozygous DNA bands, are heteroduplexes formed between amplicons harboring different indels during PCR (Zhu et al., 2014 ). The schematic diagram shows the loci at which the two sgRNAs bind. The dotted line on the PAGE image indicates the amplicon position from the wild-type allele. Gapdh , internal control. The arrow indicates the position of the PCR band obtained from the wild-type allele. (C) Calculation of editing rates ( e ) in the Ctr9 and Brca2 CRISPR embryos. The fractions of wild-type alleles (blue) and indel alleles (orange) are calculated based on the number of marks in the lanes (B) . (D) Box plot for comparison of the Ctr9 editing rates in 4-cell and 8-cell embryos. Mean values are denoted by X. (E) Summary of derivation of embryonic stem cell (ESCs) lines from the Ctr9 and Brca2 CRISPR-edited zygotes. (F) Genotyping of individual colonies ( n = 9) in each of the ESC lines (ESC1–6). Some of the ESC5 and ESC6 colonies yielded no amplicons, suggesting that these colonies may originate from the “x”-marked blastomeres that experienced large indels.
Figure Legend Snippet: Indel patterns and frequencies in mouse 8-cell embryos and embryonic stem cells derived from CRISP/Cas9-mediated editing of mouse zygotes. (A) Strategy for PCR genotyping. Eight-cell embryos derived from the microinjection of sgRNAs and recombinant Cas9 proteins into the pronuclei of mouse zygotes were disassembled to obtain single blastomeres before primary PCR to simultaneously detect the Gapdh and either the Ctr9 or the Brca2 loci. Nested or hemi-nested PCR (2° PCR) was performed using the primary PCR product, and the resulting product was combined and resolved on a polyacrylamide gel (PAGE). (B) Determination of the Ctr9 (left) and Brca2 (right) genotypes in individual blastomeres of 8-cell embryos (A to I, n = 9) based on the sizes of PCR products. Each PAGE represents a single embryo containing eight blastomeres (numbered 1-8); individual lanes are marked by “v” and “a” based on the types of alleles they contain (indel and normal, respectively). Lanes devoid of Ctr9 or Brca2 PCR product are either marked by “x” or unmarked depending on the presence or absence of the Gapdh PCR band, respectively. The blastomeres in the Ctr9 “B” embryo exhibited more PCR bands than would be expected from a diploid cell, hinting at a polyploid (Angell et al., 1987 ; Munne et al., 1994 ). The additional PCR bands at higher positions (for instance, those in the B3, B6, D5, F3, F4, H1, H3, and I6 Ctr9 embryos), especially in the blastomeres carrying heterozygous DNA bands, are heteroduplexes formed between amplicons harboring different indels during PCR (Zhu et al., 2014 ). The schematic diagram shows the loci at which the two sgRNAs bind. The dotted line on the PAGE image indicates the amplicon position from the wild-type allele. Gapdh , internal control. The arrow indicates the position of the PCR band obtained from the wild-type allele. (C) Calculation of editing rates ( e ) in the Ctr9 and Brca2 CRISPR embryos. The fractions of wild-type alleles (blue) and indel alleles (orange) are calculated based on the number of marks in the lanes (B) . (D) Box plot for comparison of the Ctr9 editing rates in 4-cell and 8-cell embryos. Mean values are denoted by X. (E) Summary of derivation of embryonic stem cell (ESCs) lines from the Ctr9 and Brca2 CRISPR-edited zygotes. (F) Genotyping of individual colonies ( n = 9) in each of the ESC lines (ESC1–6). Some of the ESC5 and ESC6 colonies yielded no amplicons, suggesting that these colonies may originate from the “x”-marked blastomeres that experienced large indels.

Techniques Used: Derivative Assay, Polymerase Chain Reaction, Recombinant, Nested PCR, Polyacrylamide Gel Electrophoresis, Amplification, CRISPR

22) Product Images from "A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)"

Article Title: A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

Journal: Heredity

doi: 10.1038/s41437-018-0070-5

Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers
Figure Legend Snippet: Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

Techniques Used: Amplification, Sequencing, Gel Extraction, Polymerase Chain Reaction

23) Product Images from "Periodic and Spatial Spreading of Alkanes and Alcanivorax Bacteria in Deep Waters of the Mariana Trench"

Article Title: Periodic and Spatial Spreading of Alkanes and Alcanivorax Bacteria in Deep Waters of the Mariana Trench

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02089-18

Global distribution of Alcanivorax in deep sea layers. The results were based on BLASTN searches of 16S rRNA fragments in 16S rRNA amplicons and metagenomes from public databases. The solid red lines show the locations of subduction zones. The map was created with Generic Mapping Tools (GMT) ( 66 ).
Figure Legend Snippet: Global distribution of Alcanivorax in deep sea layers. The results were based on BLASTN searches of 16S rRNA fragments in 16S rRNA amplicons and metagenomes from public databases. The solid red lines show the locations of subduction zones. The map was created with Generic Mapping Tools (GMT) ( 66 ).

Techniques Used:

Vertical profiling of Alcanivorax , copy numbers of functional genes, and amounts of alkanes in water samples. The abundance of Alcanivorax is an estimate based on sequencing of 16S rRNA gene amplicons. The copy numbers of functional almA and alkB genes of Alcanivorax along the water column were inferred from qPCR results. GC-MS was used to detect alkanes in the water samples. Alkanes were categorized into medium-chain (C 11 to C 16 ) and long-chain (C 17 to C 32 ) groups (see Table S2 in the supplemental material for details).
Figure Legend Snippet: Vertical profiling of Alcanivorax , copy numbers of functional genes, and amounts of alkanes in water samples. The abundance of Alcanivorax is an estimate based on sequencing of 16S rRNA gene amplicons. The copy numbers of functional almA and alkB genes of Alcanivorax along the water column were inferred from qPCR results. GC-MS was used to detect alkanes in the water samples. Alkanes were categorized into medium-chain (C 11 to C 16 ) and long-chain (C 17 to C 32 ) groups (see Table S2 in the supplemental material for details).

Techniques Used: Functional Assay, Sequencing, Real-time Polymerase Chain Reaction, Gas Chromatography-Mass Spectrometry

Collection of samples from the Mariana Trench. The water samples were collected with Niskin bottles (yellow dots) and a manned submersible (red stars) in two cruises (R/V TS01 and DY37II ) (A). Multiple sampling sites were selected to illustrate the vertical distribution of Alcanivorax . Both latitude (B) and longitude (C) distributions are depicted for the sites of the DY37II cruise, and the latitude distribution is shown for the samples collected in the TS01 cruise (D). The percentages of Alcanivorax in the mesopelagic and abyssal layers (500 to 6,000 m) were based on sequencing of the 16S rRNA gene amplicons ( 65 ). The inset map was created with Generic Mapping Tools (GMT) ( 66 ).
Figure Legend Snippet: Collection of samples from the Mariana Trench. The water samples were collected with Niskin bottles (yellow dots) and a manned submersible (red stars) in two cruises (R/V TS01 and DY37II ) (A). Multiple sampling sites were selected to illustrate the vertical distribution of Alcanivorax . Both latitude (B) and longitude (C) distributions are depicted for the sites of the DY37II cruise, and the latitude distribution is shown for the samples collected in the TS01 cruise (D). The percentages of Alcanivorax in the mesopelagic and abyssal layers (500 to 6,000 m) were based on sequencing of the 16S rRNA gene amplicons ( 65 ). The inset map was created with Generic Mapping Tools (GMT) ( 66 ).

Techniques Used: Sampling, Sequencing

Relative abundance of Alcanivorax 16S rRNA in transcripts and amplicons. The 16S rRNA gene transcripts in seven metatranscriptomes were extracted and classified. The percentages assigned to A. jadensis among all of the 16S rRNA gene transcripts are shown. The relative abundance of A. jadensis in the microbial communities of these samples was estimated based on the percentage in sequenced 16S rRNA gene amplicons.
Figure Legend Snippet: Relative abundance of Alcanivorax 16S rRNA in transcripts and amplicons. The 16S rRNA gene transcripts in seven metatranscriptomes were extracted and classified. The percentages assigned to A. jadensis among all of the 16S rRNA gene transcripts are shown. The relative abundance of A. jadensis in the microbial communities of these samples was estimated based on the percentage in sequenced 16S rRNA gene amplicons.

Techniques Used:

24) Product Images from "Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves"

Article Title: Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves

Journal: eLife

doi: 10.7554/eLife.23156

Cloning strategy. ( A ) The iRA11 amplicon library, which was prepared from microarray-synthesized oligos containing variant CDR1H or variant CDR3H regions. This amplicon is flanked by inward-facing BsaI restriction sites. ( B ) The pRA10 cloning vector, which contains the ccdB selection gene within a cassette flanked by outward-facing BsmBI restriction sites. ( C ) The pRA11 plasmid library, which was cloned by ligating BsaI-digested iRA11 amplicons and BsmBI-digest pRA10 vector. ( D ) The sequencing amplicon that was amplified from sorted cells after Tite-Seq and Sort-Seq experiments and submitted for ultra-high-throughput DNA sequencing. Appendix 3 provides more details about iRA11 amplicons, the pRA10 vector, and the pRA11 plasmid library. Appendix 4 provides more information about the creation of sequencing amplicons. DOI: http://dx.doi.org/10.7554/eLife.23156.005
Figure Legend Snippet: Cloning strategy. ( A ) The iRA11 amplicon library, which was prepared from microarray-synthesized oligos containing variant CDR1H or variant CDR3H regions. This amplicon is flanked by inward-facing BsaI restriction sites. ( B ) The pRA10 cloning vector, which contains the ccdB selection gene within a cassette flanked by outward-facing BsmBI restriction sites. ( C ) The pRA11 plasmid library, which was cloned by ligating BsaI-digested iRA11 amplicons and BsmBI-digest pRA10 vector. ( D ) The sequencing amplicon that was amplified from sorted cells after Tite-Seq and Sort-Seq experiments and submitted for ultra-high-throughput DNA sequencing. Appendix 3 provides more details about iRA11 amplicons, the pRA10 vector, and the pRA11 plasmid library. Appendix 4 provides more information about the creation of sequencing amplicons. DOI: http://dx.doi.org/10.7554/eLife.23156.005

Techniques Used: Clone Assay, Amplification, Microarray, Synthesized, Variant Assay, Plasmid Preparation, Selection, Sequencing, High Throughput Screening Assay, DNA Sequencing

25) Product Images from "Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs"

Article Title: Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00701

Primer location and amplicon coverage. Schematic view of the SLA class I gene and the location of primers used for generation of amplicons to be sequenced (primer sequences are shown in Table 1 ).
Figure Legend Snippet: Primer location and amplicon coverage. Schematic view of the SLA class I gene and the location of primers used for generation of amplicons to be sequenced (primer sequences are shown in Table 1 ).

Techniques Used: Amplification

26) Product Images from "Bead-linked transposomes enable a normalization-free workflow for NGS library preparation"

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

Journal: BMC Genomics

doi: 10.1186/s12864-018-5096-9

Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum
Figure Legend Snippet: Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum

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

Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region
Figure Legend Snippet: Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region

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

27) Product Images from "Public antibodies to malaria antigens generated by two LAIR1insertion modalities"

Article Title: Public antibodies to malaria antigens generated by two LAIR1insertion modalities

Journal: Nature

doi: 10.1038/nature23670

Validation of switch region inserts combining Illumina and MinION technologies. a, Illumina and MinION workflows. Switch regions of polyclonal naïve or IgG/IgA switched B cells were amplified by PCR. For Illumina sequencing, PCR amplicons were fragmented, re-amplified during library preparation and sequenced using the 2x300 bp MiSeq system. The bioinformatic analysis included the assembly of contiguous, chimeric reads. For insert confirmation, independently generated PCR-barcoded primary products were sequenced with MinION technology and analyzed with a different bioinformatic approach for long, error-prone MinION reads. b , Multiple identical switch inserts for donor 6 were confirmed in biological replicate experiments with independent technical and analytical setups. Shown are the experimental designs, shared and unique reads in a Venn diagram and an alignment of Illumina and MinION sequences covering the switch insertion sites for two examples ( LCP1, RAVER1 ). c , Shared and unique switch inserts in technical and biological replicate experiments of donor 5.
Figure Legend Snippet: Validation of switch region inserts combining Illumina and MinION technologies. a, Illumina and MinION workflows. Switch regions of polyclonal naïve or IgG/IgA switched B cells were amplified by PCR. For Illumina sequencing, PCR amplicons were fragmented, re-amplified during library preparation and sequenced using the 2x300 bp MiSeq system. The bioinformatic analysis included the assembly of contiguous, chimeric reads. For insert confirmation, independently generated PCR-barcoded primary products were sequenced with MinION technology and analyzed with a different bioinformatic approach for long, error-prone MinION reads. b , Multiple identical switch inserts for donor 6 were confirmed in biological replicate experiments with independent technical and analytical setups. Shown are the experimental designs, shared and unique reads in a Venn diagram and an alignment of Illumina and MinION sequences covering the switch insertion sites for two examples ( LCP1, RAVER1 ). c , Shared and unique switch inserts in technical and biological replicate experiments of donor 5.

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

28) Product Images from "Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters"

Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00830

Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
Figure Legend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

Techniques Used: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

29) Product Images from "Saturating Mutagenesis of an Essential Gene: a Majority of the Neisseria gonorrhoeae Major Outer Membrane Porin (PorB) Is Mutable"

Article Title: Saturating Mutagenesis of an Essential Gene: a Majority of the Neisseria gonorrhoeae Major Outer Membrane Porin (PorB) Is Mutable

Journal: Journal of Bacteriology

doi: 10.1128/JB.01073-13

Sequencing strategy for identifying mutable residues in PorB. Mutated porin sequences were amplified from input plasmid and output genomic DNA pools to generate 200-bp-long amplicons offset by 50 nt along the length of the porB gene. Each amplicon contained both mutated and unmutated sequences (represented by colored and black line segments, respectively), and amplifications were done in triplicate. After purification and quantitation, the amplicons were pooled and ligated to Illumina TruSeq and indexed adapters (represented by the gray line segments) for sequencing. Sequences in the reverse orientation were discarded in the data analysis, with the exception of the two amplicons at the C terminus of the protein, where only the reverse complement sequence was read.
Figure Legend Snippet: Sequencing strategy for identifying mutable residues in PorB. Mutated porin sequences were amplified from input plasmid and output genomic DNA pools to generate 200-bp-long amplicons offset by 50 nt along the length of the porB gene. Each amplicon contained both mutated and unmutated sequences (represented by colored and black line segments, respectively), and amplifications were done in triplicate. After purification and quantitation, the amplicons were pooled and ligated to Illumina TruSeq and indexed adapters (represented by the gray line segments) for sequencing. Sequences in the reverse orientation were discarded in the data analysis, with the exception of the two amplicons at the C terminus of the protein, where only the reverse complement sequence was read.

Techniques Used: Sequencing, Amplification, Plasmid Preparation, Purification, Quantitation Assay

30) Product Images from "Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement"

Article Title: Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

Journal: BMC Biology

doi: 10.1186/s12915-019-0711-z

Experimental design. a Overview of the experimental approach showing CRISPR dual cuts for removing and replacing the HPRT1 CpG island with an in vitro methylated DNA sequence through NHEJ-mediated repair. b The HPRT1 CpG island was cloned, and synonymous coding SNVs were introduced to create two distinguishable alleles (blue and purple). Cloned CpG island alleles were PCR amplified for linearization and to incorporate PAM mutations. Portions of the resulting amplicons were in vitro methylated (cyan) with M.SssI. c For each replicate, the methylated version of one allele amplicon and the unmethylated version of the other allele amplicon, together with plasmids bearing Cas9-2A-GFP and two gRNAs, were co-transfected into Hap1 cells. In one plate of Hap1 cells, allele 1 was methylated and allele 2 was not, and in a parallel experiment, allele 2 was methylated and allele 1 was not. Transfected cells were sorted by FACS and re-plated for genome editing. Edited cells were then either selected with 6-TG, which will select for cells that do not express HPRT1 , or mock selected with DMSO. Cells were harvested before and after selection, DNA was extracted, and the relevant regions PCR amplified and sequenced. The alleles allow tracking of the inserted methylated vs. unmethylated CpG island amplicons without requiring bisulfite conversion. The relative frequencies of the methylated and unmethylated alleles were calculated and compared between the 6-TG-selected, mock-selected, and pre-selection cells. d Potential outcomes of genome editing are shown for a hypothetical single cell from a single replicate. After a CRISPR dual cut, the possible outcomes at the DNA level are a deletion of the CpG island, re-insertion of the original wild-type CpG island that was cut out, or insertion of the methylated or unmethylated alleles that were transfected in. Inserted CpG islands can be inserted in an inverted or forward orientation. HPRT1 will be expressed if either the original wild-type or the unmethylated allele is inserted, but will no longer be expressed if a deletion or inversion occurs. Insertion of a forward-oriented, methylated allele should result in methylation-induced silencing. Finally, cells are expected to survive 6-TG selection if they no longer express HPRT1 , which can be a consequence of methylation-induced silencing, deletion of the CpG island, or inversion of the CpG island. Therefore, upon sequencing after 6-TG selection, if the methylated allele is inserted, we predicted that its relative frequency will be increased as compared to the unmethylated allele.
Figure Legend Snippet: Experimental design. a Overview of the experimental approach showing CRISPR dual cuts for removing and replacing the HPRT1 CpG island with an in vitro methylated DNA sequence through NHEJ-mediated repair. b The HPRT1 CpG island was cloned, and synonymous coding SNVs were introduced to create two distinguishable alleles (blue and purple). Cloned CpG island alleles were PCR amplified for linearization and to incorporate PAM mutations. Portions of the resulting amplicons were in vitro methylated (cyan) with M.SssI. c For each replicate, the methylated version of one allele amplicon and the unmethylated version of the other allele amplicon, together with plasmids bearing Cas9-2A-GFP and two gRNAs, were co-transfected into Hap1 cells. In one plate of Hap1 cells, allele 1 was methylated and allele 2 was not, and in a parallel experiment, allele 2 was methylated and allele 1 was not. Transfected cells were sorted by FACS and re-plated for genome editing. Edited cells were then either selected with 6-TG, which will select for cells that do not express HPRT1 , or mock selected with DMSO. Cells were harvested before and after selection, DNA was extracted, and the relevant regions PCR amplified and sequenced. The alleles allow tracking of the inserted methylated vs. unmethylated CpG island amplicons without requiring bisulfite conversion. The relative frequencies of the methylated and unmethylated alleles were calculated and compared between the 6-TG-selected, mock-selected, and pre-selection cells. d Potential outcomes of genome editing are shown for a hypothetical single cell from a single replicate. After a CRISPR dual cut, the possible outcomes at the DNA level are a deletion of the CpG island, re-insertion of the original wild-type CpG island that was cut out, or insertion of the methylated or unmethylated alleles that were transfected in. Inserted CpG islands can be inserted in an inverted or forward orientation. HPRT1 will be expressed if either the original wild-type or the unmethylated allele is inserted, but will no longer be expressed if a deletion or inversion occurs. Insertion of a forward-oriented, methylated allele should result in methylation-induced silencing. Finally, cells are expected to survive 6-TG selection if they no longer express HPRT1 , which can be a consequence of methylation-induced silencing, deletion of the CpG island, or inversion of the CpG island. Therefore, upon sequencing after 6-TG selection, if the methylated allele is inserted, we predicted that its relative frequency will be increased as compared to the unmethylated allele.

Techniques Used: CRISPR, In Vitro, Methylation, Sequencing, Non-Homologous End Joining, Clone Assay, Polymerase Chain Reaction, Amplification, Transfection, FACS, Selection

31) Product Images from "Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus"

Article Title: Full-Genome Deep Sequencing and Phylogenetic Analysis of Novel Human Betacoronavirus

Journal: Emerging Infectious Diseases

doi: 10.3201/eid1905.130057

A) Primers designed for reverse transcription and overlapping PCR amplification of the novel coronavirus (CoV). Dots indicate the predicted binding site of each primer along the EMC/2012 genome (x-axis). Gray bars indicate predicted amplicon lengths. Amplicon numbers are indicated beside each set of products. B) PCR products (3 µL of a 25-µL reaction) were resolved by electrophoresis on a 0.6% agarose gel and visualized by ethidium bromide staining. Lane M is the molecular weight marker (sizes indicated at left), Lanes 1–15 show the products of the amplicons depicted in Panel A. Lane C is the reagent PCR control.
Figure Legend Snippet: A) Primers designed for reverse transcription and overlapping PCR amplification of the novel coronavirus (CoV). Dots indicate the predicted binding site of each primer along the EMC/2012 genome (x-axis). Gray bars indicate predicted amplicon lengths. Amplicon numbers are indicated beside each set of products. B) PCR products (3 µL of a 25-µL reaction) were resolved by electrophoresis on a 0.6% agarose gel and visualized by ethidium bromide staining. Lane M is the molecular weight marker (sizes indicated at left), Lanes 1–15 show the products of the amplicons depicted in Panel A. Lane C is the reagent PCR control.

Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

32) Product Images from "Variation in mitochondrial minichromosome composition between blood-sucking lice of the genus Haematopinus that infest horses and pigs"

Article Title: Variation in mitochondrial minichromosome composition between blood-sucking lice of the genus Haematopinus that infest horses and pigs

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-7-144

PCR amplicons from the mitochondrial genome of the horse louse, Haematopinus asini . (A) Amplicons generated with the horse-louse-specific primers, 12sB2448F–12sB2448R (lane 2), 16sB2448F–16sB2448R (lane 3), cox1B2448F–cox1B2448R (lane 4) and cox2B2448F–cox2B2448R (lane 5) from four mitochondrial minichromosomes. Lane 1 and lane 6: 100-bp Ladder and 1-kb Ladder (BioSciences). (B) Amplicons generated with the primer pair B2448F-B2448R from the coding regions of all of the mitochondrial minichromosomes of the horse louse (lane 2). Lane 1: 500-bp DNA Ladder (Tiangen). (C) PCR verification of the mt minichromosomes of the horse louse. Lane 1 and 12: 100-bp ladder. Lane 2 and 13: 1-kb ladder. Lane 3–11: PCR amplicons from the nine minichromosomes of the horse louse: K- nad4 -atp8-atp6-N , nad2 -I-cox1-L 2 , D-Y- cox2 -S 1 -S 2 -P-cox3-A , E- cob -V , Q-nad1-T-G- nad3 -W , H- nad5 -F-nad6 , M , L 1 -rrnL and R-nad4L- rrnS -C . Genes from which PCR primers were designed are in bold.
Figure Legend Snippet: PCR amplicons from the mitochondrial genome of the horse louse, Haematopinus asini . (A) Amplicons generated with the horse-louse-specific primers, 12sB2448F–12sB2448R (lane 2), 16sB2448F–16sB2448R (lane 3), cox1B2448F–cox1B2448R (lane 4) and cox2B2448F–cox2B2448R (lane 5) from four mitochondrial minichromosomes. Lane 1 and lane 6: 100-bp Ladder and 1-kb Ladder (BioSciences). (B) Amplicons generated with the primer pair B2448F-B2448R from the coding regions of all of the mitochondrial minichromosomes of the horse louse (lane 2). Lane 1: 500-bp DNA Ladder (Tiangen). (C) PCR verification of the mt minichromosomes of the horse louse. Lane 1 and 12: 100-bp ladder. Lane 2 and 13: 1-kb ladder. Lane 3–11: PCR amplicons from the nine minichromosomes of the horse louse: K- nad4 -atp8-atp6-N , nad2 -I-cox1-L 2 , D-Y- cox2 -S 1 -S 2 -P-cox3-A , E- cob -V , Q-nad1-T-G- nad3 -W , H- nad5 -F-nad6 , M , L 1 -rrnL and R-nad4L- rrnS -C . Genes from which PCR primers were designed are in bold.

Techniques Used: Polymerase Chain Reaction, Generated

33) Product Images from "A polymerase engineered for bisulfite sequencing"

Article Title: A polymerase engineered for bisulfite sequencing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv798

PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).
Figure Legend Snippet: PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Molecular Weight

Breakdown of error rates by individual nucleotides. Data are presented as average +/−SD of the errors over the four amplicons ( prkcdbp , dab2ip , ptgs2 and ezh2 ) of bisulfite-treated genomic DNA. A breakdown of error rates by individual nucleotides for each amplicon is shown in Supplementary Tables S7 A–D. Only error rates of A, T and G are shown, as during bisulfite treatment C can be either converted to T or remain as C depending on its methylation status and the efficiency of BS conversion, which makes it impossible to assess the real error rate. For T only errors in the original Ts in the genomic DNA (no C to T conversions) are shown.
Figure Legend Snippet: Breakdown of error rates by individual nucleotides. Data are presented as average +/−SD of the errors over the four amplicons ( prkcdbp , dab2ip , ptgs2 and ezh2 ) of bisulfite-treated genomic DNA. A breakdown of error rates by individual nucleotides for each amplicon is shown in Supplementary Tables S7 A–D. Only error rates of A, T and G are shown, as during bisulfite treatment C can be either converted to T or remain as C depending on its methylation status and the efficiency of BS conversion, which makes it impossible to assess the real error rate. For T only errors in the original Ts in the genomic DNA (no C to T conversions) are shown.

Techniques Used: Amplification, Methylation

34) Product Images from "5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine"

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

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.03084-15

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

Techniques Used: Mutagenesis, Sequencing, Amplification

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

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

35) Product Images from "Mapping anaerobic sludge bed community adaptations to manure supernatant in biogas reactors"

Article Title: Mapping anaerobic sludge bed community adaptations to manure supernatant in biogas reactors

Journal: Scientific Reports

doi: 10.1038/s41598-018-34088-1

Relative abundances of bacterial classes ( A ) and archaeal genera ( B ) represented in the v3-4 16S rRNA gene amplicons obtained for individual influent, reactor, and granular samples. Each bar represents one sample. D: day; PP: pulp and paper granules; Inf HA: influent to HA reactors; Inf LA: influent to LA reactors; Cand .: Candidatus ; g.i.s .: genera_incertae_sedis . “Unclassified” indicates that OTUs could not be classified at the domain level, “Uncl” indicates that OTUs could not be classified at class level ( A ) or family level ( B ). Only taxa represented by a portion of ≥1% of the sequence reads in at least one of the samples are shown. “Others” includes all reads representing the taxa with lower abundance in all samples.
Figure Legend Snippet: Relative abundances of bacterial classes ( A ) and archaeal genera ( B ) represented in the v3-4 16S rRNA gene amplicons obtained for individual influent, reactor, and granular samples. Each bar represents one sample. D: day; PP: pulp and paper granules; Inf HA: influent to HA reactors; Inf LA: influent to LA reactors; Cand .: Candidatus ; g.i.s .: genera_incertae_sedis . “Unclassified” indicates that OTUs could not be classified at the domain level, “Uncl” indicates that OTUs could not be classified at class level ( A ) or family level ( B ). Only taxa represented by a portion of ≥1% of the sequence reads in at least one of the samples are shown. “Others” includes all reads representing the taxa with lower abundance in all samples.

Techniques Used: Sequencing

36) Product Images from "Evolutionary analysis of the Chikungunya virus epidemic in Mexico reveals intra-host mutational hotspots in the E1 protein"

Article Title: Evolutionary analysis of the Chikungunya virus epidemic in Mexico reveals intra-host mutational hotspots in the E1 protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0209292

Abundance of viral haplotypes in two E1 gene amplicons. Heat maps indicating the relative abundance (red colour intensity) of distinct amino acid viral haplotypes following deep sequencing of two amplicons COF 1 (A) and CIF 2 (B) that span the near full-length CHIKV E1 gene in 25 Mexican patients. Dendrograms represent clustering of patients according to haplotype diversity.
Figure Legend Snippet: Abundance of viral haplotypes in two E1 gene amplicons. Heat maps indicating the relative abundance (red colour intensity) of distinct amino acid viral haplotypes following deep sequencing of two amplicons COF 1 (A) and CIF 2 (B) that span the near full-length CHIKV E1 gene in 25 Mexican patients. Dendrograms represent clustering of patients according to haplotype diversity.

Techniques Used: Sequencing

37) Product Images from "A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function"

Article Title: A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function

Journal: Scientific Reports

doi: 10.1038/s41598-018-24036-4

duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.
Figure Legend Snippet: duoxa germline mutants develop goitrous thyroid phenotypes. ( A ) Zebrafish duoxa genomic locus on chromosome 25 with sequences for the wild-type (WT) allele and a mutant allele ( duoxa Δ11) containing a 11 bp deletion in exon 2. The sgRNA target site is underlined in the WT sequence. ( B ) PCR analysis of genomic DNA allows for sensitive detection of WT and duoxa Δ11 mutant alleles in individual fish (F3 generation). Polyacrylamide gel electrophoresis of PCR amplicons of WT, heterozygous and homozygous duoxa from which lanes 1, 2, 3, 4 and 6 are shown in the cropped gel image. ( C ) Thyroid phenotyping of duoxa Δ11 mutant fish maintained on a Tg ( tg:nlsEGFP ) background. Immunofluorescence staining (GFP and T4) of 6 dpf larvae (ventral view, anterior to the top, scale bars: 20 µm) showed goitrous thyroid enlargement and absence of detectable T4 staining in all homozygous duoxa Δ11 fish ( N = 30). Larvae with a normal-looking thyroid morphology ( N = 30) were genotyped as either WT or heterozygous carriers of the duoxa Δ11 allele. For each larvae shown, 3.5-fold magnified views of the thyroid region are displayed (merge of GFP/T4 and T4 only). ( D ) Proportion of larvae with goitrous thyroid phenotype as detected in the progeny of three independent inbreeding experiments with heterozygous duoxa Δ11 fish.

Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Polyacrylamide Gel Electrophoresis, Immunofluorescence, Staining

38) Product Images from "A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa"

Article Title: A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa

Journal: PLoS ONE

doi: 10.1371/journal.pone.0194527

Neoromicia adenovirus sequences. A) Overview of confirmed mastadenovirus contigs from the Neoromicia virome created with Velvet and CLC assemblers as they align to a characteristic mastadenovirus genome. The dark grey contig was used in B, along with the amplicons produced by conventional PCR (depicted by the checkered block). B) Bayesian phylogenetic tree of a 237 bp region of the DNA polymerase gene. The phylogeny was constructed in BEAST v1.8 using the Hasegawa, Kishino and Yano (HKY) substitution model plus gamma distribution model suggested by J-model test. The MCMC chain was set to 20,000,000 generations sampled every 2000 steps, with a 10% burn-in of the first generated trees. Adenovirus sequences detected from this study are shown with black circles, and bat species from which adenoviruses originated are indicated on the right side of the sequence names. Posterior probability values of less than 50% were omitted. GenBank accession numbers are shown next to sequences.
Figure Legend Snippet: Neoromicia adenovirus sequences. A) Overview of confirmed mastadenovirus contigs from the Neoromicia virome created with Velvet and CLC assemblers as they align to a characteristic mastadenovirus genome. The dark grey contig was used in B, along with the amplicons produced by conventional PCR (depicted by the checkered block). B) Bayesian phylogenetic tree of a 237 bp region of the DNA polymerase gene. The phylogeny was constructed in BEAST v1.8 using the Hasegawa, Kishino and Yano (HKY) substitution model plus gamma distribution model suggested by J-model test. The MCMC chain was set to 20,000,000 generations sampled every 2000 steps, with a 10% burn-in of the first generated trees. Adenovirus sequences detected from this study are shown with black circles, and bat species from which adenoviruses originated are indicated on the right side of the sequence names. Posterior probability values of less than 50% were omitted. GenBank accession numbers are shown next to sequences.

Techniques Used: Produced, Polymerase Chain Reaction, Blocking Assay, Construct, Generated, Sequencing

39) Product Images from "Efficient Retroelement-Mediated DNA Writing in Bacteria"

Article Title: Efficient Retroelement-Mediated DNA Writing in Bacteria

Journal: bioRxiv

doi: 10.1101/2020.02.21.958983

Strategy used to deplete unedited memory registers from dual-register amplicons and the frequency of cell-cell interactions recovered by high-throughput sequencing in the connectome mapping experiment. (A) Using restriction digestion as an alternative strategy to remove unedited registers from the PCR amplified amplicons instead of allele-specific PCR. Genomic DNA samples were purified from the parental recipient cells (MG1655 Δ recJ Δ xonA galK OFF ), as well as cultures obtained after conjugation (transconjugants) in the experiment described in Fig. 4C . The galK locus was PCR amplified from the purified genomic DNA samples and run on a 6% TBE gel before and after digestion with ClaI and AgeI enzymes (which cut unedited Register 1 and Register 2, respectively) and stained by SYBR gold. The galK amplicon obtained from the parental sample was completely digested after enzymatic digestion. In contrast, the galK amplicon obtained from the transconjugant sample was not completely digested by ClaI and AgeI. The undigested band, corresponding to edited registers, comprised ∼3.9% of the signal in this lane (measured by densitometry). (B) This band was subsequently excised, purified and Sanger-sequenced. Drops in the quality of sequencing in Register 1 and 2 indicate the presence of mixed DNA populations containing variations in these two regions in these samples. Subsequently, Illumina adaptors and barcodes were added to this undigested amplicon using an additional round of PCR and the obtained amplicon was sequenced by Illumina MiSeq (see Methods). (C) Number of unique variants (interactions) per million reads obtained from sequencing the two target registers in the genomes of recipient cells after conjugation with donor cells, as well as two randomly selected non-targeted regions within the galK amplicon (used as a negative control and to assess the rate of false-positives), for the experiment shown in Fig. 4C .
Figure Legend Snippet: Strategy used to deplete unedited memory registers from dual-register amplicons and the frequency of cell-cell interactions recovered by high-throughput sequencing in the connectome mapping experiment. (A) Using restriction digestion as an alternative strategy to remove unedited registers from the PCR amplified amplicons instead of allele-specific PCR. Genomic DNA samples were purified from the parental recipient cells (MG1655 Δ recJ Δ xonA galK OFF ), as well as cultures obtained after conjugation (transconjugants) in the experiment described in Fig. 4C . The galK locus was PCR amplified from the purified genomic DNA samples and run on a 6% TBE gel before and after digestion with ClaI and AgeI enzymes (which cut unedited Register 1 and Register 2, respectively) and stained by SYBR gold. The galK amplicon obtained from the parental sample was completely digested after enzymatic digestion. In contrast, the galK amplicon obtained from the transconjugant sample was not completely digested by ClaI and AgeI. The undigested band, corresponding to edited registers, comprised ∼3.9% of the signal in this lane (measured by densitometry). (B) This band was subsequently excised, purified and Sanger-sequenced. Drops in the quality of sequencing in Register 1 and 2 indicate the presence of mixed DNA populations containing variations in these two regions in these samples. Subsequently, Illumina adaptors and barcodes were added to this undigested amplicon using an additional round of PCR and the obtained amplicon was sequenced by Illumina MiSeq (see Methods). (C) Number of unique variants (interactions) per million reads obtained from sequencing the two target registers in the genomes of recipient cells after conjugation with donor cells, as well as two randomly selected non-targeted regions within the galK amplicon (used as a negative control and to assess the rate of false-positives), for the experiment shown in Fig. 4C .

Techniques Used: Next-Generation Sequencing, Polymerase Chain Reaction, Amplification, Purification, Conjugation Assay, Staining, Sequencing, Negative Control

40) Product Images from "Mapping the evolutionary landscape of Zika virus infection in immunocompromised mice"

Article Title: Mapping the evolutionary landscape of Zika virus infection in immunocompromised mice

Journal: bioRxiv

doi: 10.1101/839803

Transmission route impacts organ-specific diversity during ZIKV infection. Eucudean distance and Shannon entropy were calculated using sequence data frorri PCR1 amplicons. (A). Multidimensional scaling plot showing the all-versus-all Eucudean distance measurement of each organ sample. (B). Genetic distance of each sample from its corresponding stock control separated by tissue type. Data represent the median with inter-quartile range. Black dots represent outliers. Kruskal-Wallis with Dunn’s post-test, considering a type-I error. * p
Figure Legend Snippet: Transmission route impacts organ-specific diversity during ZIKV infection. Eucudean distance and Shannon entropy were calculated using sequence data frorri PCR1 amplicons. (A). Multidimensional scaling plot showing the all-versus-all Eucudean distance measurement of each organ sample. (B). Genetic distance of each sample from its corresponding stock control separated by tissue type. Data represent the median with inter-quartile range. Black dots represent outliers. Kruskal-Wallis with Dunn’s post-test, considering a type-I error. * p

Techniques Used: Transmission Assay, Infection, Sequencing

Total ZIKV single nucleotide varlant (SNV) and evolutionary analysis. The ZIKV coding region was amplified in three amplicons by PCR (bottom schematic) and each amplicon was used for deep sequencing analysis and alignment to the MR766 genome. Variants below 1% frequency were considerad background and were removed from the analysis (A). Viral variants present in the MR766 stocks that were used for mosquito feeding and needle infections. (B). Total viral variants present in mice subcutaneously infected with ZIKV MR766 (N=7). (C). ZIKV variants present in bodies of infected mosquitoes 14 days post feeding. After feeding on mice, mosquitoes were ground, RNA extracted, and ZIKV genome amplified by PCR. (D). Total viral variants present in mice infected by mosquito bite. Data represent mice from three inde-pendent experiments (N=3). (E). Schematic showing genomic locations of the MR766 minority variants corresponding to nucleotide changes to the Cambodian, Brazilian, and Puerto Rican strains. (F). Frequencies of MR766 minority variants corresponding to changes between MR766 and the Cambodian, Brazilian, and Puerto Rican strains. (G). Proportion of each nucleotide transition among the 86 variants identified to be different between the ZIKV strains.
Figure Legend Snippet: Total ZIKV single nucleotide varlant (SNV) and evolutionary analysis. The ZIKV coding region was amplified in three amplicons by PCR (bottom schematic) and each amplicon was used for deep sequencing analysis and alignment to the MR766 genome. Variants below 1% frequency were considerad background and were removed from the analysis (A). Viral variants present in the MR766 stocks that were used for mosquito feeding and needle infections. (B). Total viral variants present in mice subcutaneously infected with ZIKV MR766 (N=7). (C). ZIKV variants present in bodies of infected mosquitoes 14 days post feeding. After feeding on mice, mosquitoes were ground, RNA extracted, and ZIKV genome amplified by PCR. (D). Total viral variants present in mice infected by mosquito bite. Data represent mice from three inde-pendent experiments (N=3). (E). Schematic showing genomic locations of the MR766 minority variants corresponding to nucleotide changes to the Cambodian, Brazilian, and Puerto Rican strains. (F). Frequencies of MR766 minority variants corresponding to changes between MR766 and the Cambodian, Brazilian, and Puerto Rican strains. (G). Proportion of each nucleotide transition among the 86 variants identified to be different between the ZIKV strains.

Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, Mouse Assay, Infection

Related Articles

Polymerase Chain Reaction:

Article Title: 2.7 million samples genotyped for HLA by next generation sequencing: lessons learned
Article Snippet: .. The CyBi-Well vario system is also used to setup a subsequent secondary PCR reaction to elongate the amplicons with MIDs and sequencing adapters for Illumina sequencing. .. Amplicon pooling, secondary PCR setup and thermocycling last about 1 h. Whilst the Fluidigm workflow has remained technically the same since November 2015, the introduction of the 384 PCR workflow for low-concentration samples changed the average DNA-concentration of samples subjected to the Fluidigm workflow.

Sequencing:

Article Title: 2.7 million samples genotyped for HLA by next generation sequencing: lessons learned
Article Snippet: .. The CyBi-Well vario system is also used to setup a subsequent secondary PCR reaction to elongate the amplicons with MIDs and sequencing adapters for Illumina sequencing. .. Amplicon pooling, secondary PCR setup and thermocycling last about 1 h. Whilst the Fluidigm workflow has remained technically the same since November 2015, the introduction of the 384 PCR workflow for low-concentration samples changed the average DNA-concentration of samples subjected to the Fluidigm workflow.

Article Title: Anaerobic digestion of pig manure supernatant at high ammonia concentrations characterized by high abundances of Methanosaeta and non-euryarchaeotal archaea
Article Snippet: .. The samples analyzed in this study accounted for 36 of 116 uniquely indexed amplicons constituting the library that was sequenced on a MiSeq lane (Illumina, San Diego, CA) with v3 reagents employing 300 bp paired end reads at the Norwegian Sequencing Centre. .. PhiX library (Illumina) was blended to 50%.

Article Title: Microbial Community Composition and Functional Capacity in a Terrestrial Ferruginous, Sulfate-Depleted Mud Volcano
Article Snippet: .. Amplicons from different samples were pooled in equal quantities sufficient for sequencing on an Illumina MiSeq platform (Illumina, United States). .. 16S rRNA Gene Copy Number Quantitative PCR was used to analyze the 16S rRNA gene copy number of bacteria, archaea, and ANME-2a in the environmental samples and incubated mud slurries using a MyiQ Real-time PCR Detection System (Bio-Rad, United States).

Generated:

Article Title: Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario
Article Snippet: .. Pooled amplicons representing either 24 or 96 samples were used to prepare indexed libraries using Nextera XT and index kits as directed (Illumina) and 200 or 250 base paired end reads were generated on a MiSeq instrument. .. Reference-based assembly of the paired fastq files was achieved using the NGen programme of the DNASTAR Lasergene software (v. 11) using either a reference RRV genome (GenBank accession ) or more closely related sequences obtained as the study progressed.

Amplification:

Article Title: A method for high‐throughput production of sequence‐verified DNA libraries and strain collections
Article Snippet: .. To explore explanations for the poor representation of these probes, we selected the 49 colonies representing these probes, collectively amplified the exogenous DNA (i.e. the amplified array‐synthesized oligonucleotides that integrated into the target locus of the recipient strain), and sequenced those amplicons using MiSeq (Illumina). ..

Mutagenesis:

Article Title: Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides
Article Snippet: .. The amplicons were sequenced on an Illumina MiSeq, and a script created to extract all WT and mutant sequences and report the numbers. ..

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    Illumina Inc 16s rrna gene amplicons
    Neighbor-joining tree based on <t>16S</t> <t>rRNA</t> gene sequences showing phylogenetic relationships in the genus Geobacter . Refer to Table 2 for the major OTUs. Desulfuromonas acetoxidans was used as an outgroup. Bootstrap values (100 trials, only > 50 are shown) are indicated at branching points. The bar indicates 2% sequence divergence. Accession numbers are shown in parentheses.
    16s Rrna Gene Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc fungal amplicons
    Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate <t>amplicons</t> for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .
    Fungal Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc amplicon based pcr approach
    HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> <t>amplicon</t> approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Amplicon Based Pcr Approach, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neighbor-joining tree based on 16S rRNA gene sequences showing phylogenetic relationships in the genus Geobacter . Refer to Table 2 for the major OTUs. Desulfuromonas acetoxidans was used as an outgroup. Bootstrap values (100 trials, only > 50 are shown) are indicated at branching points. The bar indicates 2% sequence divergence. Accession numbers are shown in parentheses.

    Journal: PLoS ONE

    Article Title: Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

    doi: 10.1371/journal.pone.0077443

    Figure Lengend Snippet: Neighbor-joining tree based on 16S rRNA gene sequences showing phylogenetic relationships in the genus Geobacter . Refer to Table 2 for the major OTUs. Desulfuromonas acetoxidans was used as an outgroup. Bootstrap values (100 trials, only > 50 are shown) are indicated at branching points. The bar indicates 2% sequence divergence. Accession numbers are shown in parentheses.

    Article Snippet: The microbiomes established in each system were compared by pyrotag sequencing of 16S rRNA gene amplicons and shotgun metagenomics.

    Techniques: Sequencing

    Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .

    Journal: PLoS ONE

    Article Title: Complementary Amplicon-Based Genomic Approaches for the Study of Fungal Communities in Humans

    doi: 10.1371/journal.pone.0116705

    Figure Lengend Snippet: Organization of fungal rDNA locus and regions targeted by oligonucleotide probe and primers. Primers UNI1, UNI2, and Cspecies, and probe are used in the qPCR strategy. Primers Fseq and Rseq are used to generate amplicons for sequencing. Cgla, C . glabrata ; Ctro, C . tropicalis ; Cpar, C . parapsilosis ; Ckru, C . krusei ; Calb, C . albicans . Locus depiction is not to scale. Primer sequences are given in Table 1 .

    Article Snippet: We estimated, from a recent study [ ], that fungal amplicons generated with these primers would generally fit within the size range for 2 × 150 paired-end sequencing by the Illumina MiSeq platform.

    Techniques: Real-time Polymerase Chain Reaction, Sequencing

    HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Journal: Retrovirology

    Article Title: High-throughput profiling of point mutations across the HIV-1 genome

    doi: 10.1186/s12977-014-0124-6

    Figure Lengend Snippet: HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Article Snippet: We have also demonstrated the application of our amplicon-based PCR approach for Illumina NGS to clinical HIV-1 quasi-species populations in acute infection [ ] and achieved a higher sensitivity in identifying rare quasi-species variants as compared to published approaches using other NGS platforms.

    Techniques: Mutagenesis, Next-Generation Sequencing, Sample Prep, Polymerase Chain Reaction, Amplification, Concentration Assay