Structured Review

Pacific Biosciences amplicons
Primer design (A) and quality control presequencing (B) and postsequencing (C) . (A) Primers designed on the phagemid vector and used for single chain fragment variable (scFv) PCR amplification. The scFv (VH-LINKER-VL) length range is between ~720 and ~800 bp [variable heavy (VH) between ~350 and ~400 bp and variable light (VL) between ~320 and ~350 bp]. The linker is 53 bp including the EcoRI and XbaI sites. The PCR products are expected to be ~1,000 bp on average, including the 5′ and 3′ region and the primers. (B) Agarose gel electrophoresis of PCR products. The DNA was amplified from the AAR3 fraction and PCR products were analyzed on 1.2% (w/v) agarose gel. The band at ~1,000 bp corresponds to the expected size for scFv <t>amplicons.</t> S1, S2, S3, and S4 correspond to the samples 1, 2, 3, and 4, respectively. The Bioanalyzer trace of the four samples shows the purity of amplicons with a high-quality single peak. (C) Pacific Biosciences RS II CCS2 read length distribution using P6-C4 chemistry for 1 SMRT cell (similar results were obtained for the 15 SMRT cells). Data are based on a 1-kb size-selected scFv library using a 6 h movie.
Amplicons, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library"

Article Title: Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01796

Primer design (A) and quality control presequencing (B) and postsequencing (C) . (A) Primers designed on the phagemid vector and used for single chain fragment variable (scFv) PCR amplification. The scFv (VH-LINKER-VL) length range is between ~720 and ~800 bp [variable heavy (VH) between ~350 and ~400 bp and variable light (VL) between ~320 and ~350 bp]. The linker is 53 bp including the EcoRI and XbaI sites. The PCR products are expected to be ~1,000 bp on average, including the 5′ and 3′ region and the primers. (B) Agarose gel electrophoresis of PCR products. The DNA was amplified from the AAR3 fraction and PCR products were analyzed on 1.2% (w/v) agarose gel. The band at ~1,000 bp corresponds to the expected size for scFv amplicons. S1, S2, S3, and S4 correspond to the samples 1, 2, 3, and 4, respectively. The Bioanalyzer trace of the four samples shows the purity of amplicons with a high-quality single peak. (C) Pacific Biosciences RS II CCS2 read length distribution using P6-C4 chemistry for 1 SMRT cell (similar results were obtained for the 15 SMRT cells). Data are based on a 1-kb size-selected scFv library using a 6 h movie.
Figure Legend Snippet: Primer design (A) and quality control presequencing (B) and postsequencing (C) . (A) Primers designed on the phagemid vector and used for single chain fragment variable (scFv) PCR amplification. The scFv (VH-LINKER-VL) length range is between ~720 and ~800 bp [variable heavy (VH) between ~350 and ~400 bp and variable light (VL) between ~320 and ~350 bp]. The linker is 53 bp including the EcoRI and XbaI sites. The PCR products are expected to be ~1,000 bp on average, including the 5′ and 3′ region and the primers. (B) Agarose gel electrophoresis of PCR products. The DNA was amplified from the AAR3 fraction and PCR products were analyzed on 1.2% (w/v) agarose gel. The band at ~1,000 bp corresponds to the expected size for scFv amplicons. S1, S2, S3, and S4 correspond to the samples 1, 2, 3, and 4, respectively. The Bioanalyzer trace of the four samples shows the purity of amplicons with a high-quality single peak. (C) Pacific Biosciences RS II CCS2 read length distribution using P6-C4 chemistry for 1 SMRT cell (similar results were obtained for the 15 SMRT cells). Data are based on a 1-kb size-selected scFv library using a 6 h movie.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

2) Product Images from "Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea, et al. Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea"

Article Title: Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea, et al. Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.935

A histogram showing CCS read length distribution of 16S rRNA amplicons
Figure Legend Snippet: A histogram showing CCS read length distribution of 16S rRNA amplicons

Techniques Used:

3) Product Images from "Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion"

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

Journal: NPJ Genomic Medicine

doi: 10.1038/s41525-017-0042-3

Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
Figure Legend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

Techniques Used: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification

4) Product Images from "Analysis of plant microbe interactions in the era of next generation sequencing technologies"

Article Title: Analysis of plant microbe interactions in the era of next generation sequencing technologies

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2014.00216

Schematic presentation of the library preparation and sequencing process of the most commonly used next generation sequencing platforms . All different types of starting molecules are converted into doublestranded DNA molecules that are flanked by adapters. Adapters are sequencing platform specific and enable the binding of the library molecules to surfaces, either beads or a flow cell, where they are amplified prior to sequencing. Clonal amplicons are spatially separated on the glass slides, chips, or picotiterplate. Sequencing is either a sequencing by ligation process with fluorescently labeled oligonucleotides of known sequence (SOLiD) or a sequencing by synthesis process. During Illumina sequencing, four differently labeled nucleotides are flushed over the flow cell in multiple cycles, depending on the desired read length. During 454 and Ion PGM sequencing unlabeled nucleotides are flushed in a sequential order over the flow cell. Incorporation is detected via a coupled light reaction (454) or the detection of proton release during nucleotide incorporation.
Figure Legend Snippet: Schematic presentation of the library preparation and sequencing process of the most commonly used next generation sequencing platforms . All different types of starting molecules are converted into doublestranded DNA molecules that are flanked by adapters. Adapters are sequencing platform specific and enable the binding of the library molecules to surfaces, either beads or a flow cell, where they are amplified prior to sequencing. Clonal amplicons are spatially separated on the glass slides, chips, or picotiterplate. Sequencing is either a sequencing by ligation process with fluorescently labeled oligonucleotides of known sequence (SOLiD) or a sequencing by synthesis process. During Illumina sequencing, four differently labeled nucleotides are flushed over the flow cell in multiple cycles, depending on the desired read length. During 454 and Ion PGM sequencing unlabeled nucleotides are flushed in a sequential order over the flow cell. Incorporation is detected via a coupled light reaction (454) or the detection of proton release during nucleotide incorporation.

Techniques Used: Sequencing, Next-Generation Sequencing, Binding Assay, Flow Cytometry, Amplification, Ligation, Labeling

5) Product Images from "Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis"

Article Title: Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007761

Categories of sequencing reads inferred from the PacBio NGS sequencing. (A) Categories of sequencing reads deriving from the gapC amplicon, (B) i6-tubA amplicon and (C) i5-tubA amplicon, respectively. Number and percentage of particular types of reads are given. The distribution of introns in all groups of reads is depicted. Lines represent the exons while circles represent the introns: black circles: conventional introns; white: nonconventional ones (not to scale). Introns are labelled i1 –i6. In addition, introns that exhibit intermediate features are marked with an asterisk. Double asterisk indicates the introns to which the reverse primer was targeted, thus not considered in the analysis of amplicons (introns to which primers annealed were inevitably present in the amplification products).
Figure Legend Snippet: Categories of sequencing reads inferred from the PacBio NGS sequencing. (A) Categories of sequencing reads deriving from the gapC amplicon, (B) i6-tubA amplicon and (C) i5-tubA amplicon, respectively. Number and percentage of particular types of reads are given. The distribution of introns in all groups of reads is depicted. Lines represent the exons while circles represent the introns: black circles: conventional introns; white: nonconventional ones (not to scale). Introns are labelled i1 –i6. In addition, introns that exhibit intermediate features are marked with an asterisk. Double asterisk indicates the introns to which the reverse primer was targeted, thus not considered in the analysis of amplicons (introns to which primers annealed were inevitably present in the amplification products).

Techniques Used: Sequencing, Next-Generation Sequencing, Amplification

6) Product Images from "Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing"

Article Title: Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117050

IGHA 3′ UTR contains novel splice junction. a) Full length cDNA of single B cells was generated using the C1 autoprep system, fragmented using Nextera XT, and sequenced using Illumina sequencers (RNAseq). Additionally, RNA was extracted from bulk B cells and the IGH transcripts were analyzed using the PacBio sequencer (IGH-Seq) or conventional Sanger sequencing after gel isolation b) RNAseq reads of two B cells were aligned to the IGH locus. Coverage density is shown as a histogram for both IGHA1 and IGHM exons for both B cells. Coverage density in the IGHA1 expressing cell indicated a splicing event in the canonical IGHA membrane exon. c) PacBio single molecule sequencing reads were mapped to the IGHA1 locus. Reads containing the whole VDJ region as well as either S or M exons were grouped and quantified. This confirmed the presence of a splice site in the canonical IGHA1 membrane exon resulting in two exons (named IGHA1 M1 and M2). d) Gel separation of amplicons generated from bulk B cell RNA using primers specific for exon J4 and putative exon IGHA1 M2 on the left. Schematic representation of isoform splice structure on the right. The longer band confirmed the IGHA1 M1 to M2 splicing event, the shorter band represents a novel isoform of IGHA1.
Figure Legend Snippet: IGHA 3′ UTR contains novel splice junction. a) Full length cDNA of single B cells was generated using the C1 autoprep system, fragmented using Nextera XT, and sequenced using Illumina sequencers (RNAseq). Additionally, RNA was extracted from bulk B cells and the IGH transcripts were analyzed using the PacBio sequencer (IGH-Seq) or conventional Sanger sequencing after gel isolation b) RNAseq reads of two B cells were aligned to the IGH locus. Coverage density is shown as a histogram for both IGHA1 and IGHM exons for both B cells. Coverage density in the IGHA1 expressing cell indicated a splicing event in the canonical IGHA membrane exon. c) PacBio single molecule sequencing reads were mapped to the IGHA1 locus. Reads containing the whole VDJ region as well as either S or M exons were grouped and quantified. This confirmed the presence of a splice site in the canonical IGHA1 membrane exon resulting in two exons (named IGHA1 M1 and M2). d) Gel separation of amplicons generated from bulk B cell RNA using primers specific for exon J4 and putative exon IGHA1 M2 on the left. Schematic representation of isoform splice structure on the right. The longer band confirmed the IGHA1 M1 to M2 splicing event, the shorter band represents a novel isoform of IGHA1.

Techniques Used: Generated, Sequencing, Isolation, Expressing

Alternative splicing of IGHM transcripts. a) RNA was extracted from bulk B cells and the IGH transcripts were analyzed using the PacBio sequencer (IGH-Seq) and mapped to the IGHM locus. Reads containing the whole VDJ region as well as either S or M exons were grouped and quantified. Several reads represented abundant novel short IGHM isoforms lacking structural exons. b) Gel separation of amplicons generated from bulk B cell RNA using primers specific for exon J4 and putative exon IGHA M2 is shown on theleft. Schematic representation of isoform splice structure as validated by Sanger sequencing is shown on the right. The bands of several sizes confirmed the presence of short IGHM isoforms found by single molecule sequencing. c) Mutation rates of different IGH isoforms derived from PacBio reads shown as boxplots. The low mutation rates of the novel isotypes strongly indicate that their expression is limited to naïve B cells.
Figure Legend Snippet: Alternative splicing of IGHM transcripts. a) RNA was extracted from bulk B cells and the IGH transcripts were analyzed using the PacBio sequencer (IGH-Seq) and mapped to the IGHM locus. Reads containing the whole VDJ region as well as either S or M exons were grouped and quantified. Several reads represented abundant novel short IGHM isoforms lacking structural exons. b) Gel separation of amplicons generated from bulk B cell RNA using primers specific for exon J4 and putative exon IGHA M2 is shown on theleft. Schematic representation of isoform splice structure as validated by Sanger sequencing is shown on the right. The bands of several sizes confirmed the presence of short IGHM isoforms found by single molecule sequencing. c) Mutation rates of different IGH isoforms derived from PacBio reads shown as boxplots. The low mutation rates of the novel isotypes strongly indicate that their expression is limited to naïve B cells.

Techniques Used: Generated, Sequencing, Mutagenesis, Derivative Assay, Expressing

7) Product Images from "Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries"

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

Journal: BMC Bioinformatics

doi: 10.1186/s12859-018-2293-0

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

Techniques Used: Sequencing

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

Techniques Used: Sequencing

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

Techniques Used: Sequencing, Generated, Amplification

8) Product Images from "Dynamics of coral‐associated microbiomes during a thermal bleaching event, et al. Dynamics of coral‐associated microbiomes during a thermal bleaching event"

Article Title: Dynamics of coral‐associated microbiomes during a thermal bleaching event, et al. Dynamics of coral‐associated microbiomes during a thermal bleaching event

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.604

A bar chart illustrating the percentages of 16S rRNA sequence reads that are classifiable at the species level, using full‐length (blue) or partial amplicons (V3‐V4, red; V5‐V6, green)
Figure Legend Snippet: A bar chart illustrating the percentages of 16S rRNA sequence reads that are classifiable at the species level, using full‐length (blue) or partial amplicons (V3‐V4, red; V5‐V6, green)

Techniques Used: Sequencing

9) Product Images from "Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue"

Article Title: Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue

Journal: Scientific Reports

doi: 10.1038/s41598-018-20623-7

Phasing of de novo variants with variants known to belong to either the wildtype (MCF-7) or mutant (MDA-MB-231) genotype, exemplified by four Integrative Genomics Viewer (IGV) screenshots. ( A ) In the 0.1% mutant sample, position 7029 (T, red) phases together with reads containing the wildtype (MCF-7) variant at position 6776 (C, blue) but not the mutant (MDA-MB-231) variants at positions 7028 (T, red) and 8506 (C, blue). ( B ) In the 10% mutant sample, position 10406 (A, green) phases together with reads containing the mutant (MDA-MB-231) variants at position 11719 (A, green) and 12084 (T, red) but not the wildtype (MCF-7) variant at position 9966 (A, green). Note that position 10406 is covered by two amplicons, and thus detected by two independent observations. ( C ) In the 10% mutant sample, position 13623 (T, red) phases together with reads containing the mutant (MDA-MB-231) variants at position 12705 (T, red), 13966 (G, orange), 14470 (C, blue), 14766 (T, red) and 15310 (C, blue) but not the wildtype (MCF-7) variants at position 13260 (C, blue) and 14319 (C, blue). Note that position 13623 is covered by two amplicons, and thus detected by two independent observations. ( D ) In the 0.1% mutant sample, position 15897 (A, green) phases together with reads containing the wildtype (MCF-7) variants at position 15380 (G, orange) and 16148 (T, red) but not the mutant (MDA-MB-231) variants at position 15310 (C, blue), 16093 (C, blue), 16184 (A, green), 16189 (C, blue), 16223 (T, red), 16265 (G, orange) and 16278 (T, red). Horizontal is the DNA sequence, vertical the individual reads, and alignments sorted by base. Note that the position in IGV does not correspond to the rCRS position due to the use of an extended reference for alignment (see Materials and Methods). INDELs
Figure Legend Snippet: Phasing of de novo variants with variants known to belong to either the wildtype (MCF-7) or mutant (MDA-MB-231) genotype, exemplified by four Integrative Genomics Viewer (IGV) screenshots. ( A ) In the 0.1% mutant sample, position 7029 (T, red) phases together with reads containing the wildtype (MCF-7) variant at position 6776 (C, blue) but not the mutant (MDA-MB-231) variants at positions 7028 (T, red) and 8506 (C, blue). ( B ) In the 10% mutant sample, position 10406 (A, green) phases together with reads containing the mutant (MDA-MB-231) variants at position 11719 (A, green) and 12084 (T, red) but not the wildtype (MCF-7) variant at position 9966 (A, green). Note that position 10406 is covered by two amplicons, and thus detected by two independent observations. ( C ) In the 10% mutant sample, position 13623 (T, red) phases together with reads containing the mutant (MDA-MB-231) variants at position 12705 (T, red), 13966 (G, orange), 14470 (C, blue), 14766 (T, red) and 15310 (C, blue) but not the wildtype (MCF-7) variants at position 13260 (C, blue) and 14319 (C, blue). Note that position 13623 is covered by two amplicons, and thus detected by two independent observations. ( D ) In the 0.1% mutant sample, position 15897 (A, green) phases together with reads containing the wildtype (MCF-7) variants at position 15380 (G, orange) and 16148 (T, red) but not the mutant (MDA-MB-231) variants at position 15310 (C, blue), 16093 (C, blue), 16184 (A, green), 16189 (C, blue), 16223 (T, red), 16265 (G, orange) and 16278 (T, red). Horizontal is the DNA sequence, vertical the individual reads, and alignments sorted by base. Note that the position in IGV does not correspond to the rCRS position due to the use of an extended reference for alignment (see Materials and Methods). INDELs

Techniques Used: Mutagenesis, Multiple Displacement Amplification, Variant Assay, Sequencing

10) Product Images from "A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium"

Article Title: A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium

Journal: mBio

doi: 10.1128/mBio.02354-17

Schematic overview of the PacBio barcoding approach. For simplicity, this schematic shows how amplicon libraries are created from two separate samples. The approach includes two PCRs, the first specific to the target with M13 motifs on the 5′ and 3′ ends of the forward and reverse primers, respectively. The second PCR uses the product from step 1 and involves the addition of unique forward and reverse barcodes to the amplicons. Overall, the amplicon library created for each sample possesses a unique combination of barcodes, allowing both sample libraries to be pooled and distinguished postsequencing. Upon pooling, equal concentrations of the amplicon libraries (step 4) are submitted for PacBio sequencing by the CCS approach entailing circularization of the amplicons (SMRTbell).
Figure Legend Snippet: Schematic overview of the PacBio barcoding approach. For simplicity, this schematic shows how amplicon libraries are created from two separate samples. The approach includes two PCRs, the first specific to the target with M13 motifs on the 5′ and 3′ ends of the forward and reverse primers, respectively. The second PCR uses the product from step 1 and involves the addition of unique forward and reverse barcodes to the amplicons. Overall, the amplicon library created for each sample possesses a unique combination of barcodes, allowing both sample libraries to be pooled and distinguished postsequencing. Upon pooling, equal concentrations of the amplicon libraries (step 4) are submitted for PacBio sequencing by the CCS approach entailing circularization of the amplicons (SMRTbell).

Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing

11) Product Images from "Detecting PKD1 variants in polycystic kidney disease patients by single‐molecule long‐read sequencing, et al. Detecting PKD1 variants in polycystic kidney disease patients by single‐molecule long‐read sequencing"

Article Title: Detecting PKD1 variants in polycystic kidney disease patients by single‐molecule long‐read sequencing, et al. Detecting PKD1 variants in polycystic kidney disease patients by single‐molecule long‐read sequencing

Journal: Human Mutation

doi: 10.1002/humu.23223

SMRT sequencing and variant calling of LR‐PCR amplicons. A: Sequencing depth (DP; in number of reads) of the alignments to chromosome 16 and chromosome 4. Number of uniquely aligned reads (y axis, blue line) sequenced with PacBio that mapped to PKD1 and PKD2 . Off‐target amplification is discriminated from the main PKD1 gene sequences showing alignments to pseudogene homologous sequences at proximal loci (e.g., PKD1P1 , PKD1P5 , PKD1P6 ) (blue boxes). B: Mapping quality (MQ; in Phred quality scores; values > 90 were scaled down for visualization purposes), and sequencing depth (DP; in number of reads) of uniquely aligned molecules to PKD1 (NM_001009944.2) for the five LR‐PCR fragments amplified. Mapping quality of alignments with even coverage distribution along the amplified fragments (fragments), including regions with SDs, repetitive elements (repeats), and high GC content (GC%). Despite fragments A and E showing lower coverage, compared with the average sequencing depth of ≥421× (minimum ≥19×; maximum 1,528×), they had sufficient coverage for variant calling within the exon regions, including the first exons of PKD1 , with average coverage of ≥55× (minimum ≥24×; maximum 91×) (Supp. Table S4). C : We detected 1,506 intron variants (blue) and 177 coding or splice‐site variants (yellow). The predicted transcript effects of coding and splice‐site variants were quantified (bar chart) as log10 count (x axis)
Figure Legend Snippet: SMRT sequencing and variant calling of LR‐PCR amplicons. A: Sequencing depth (DP; in number of reads) of the alignments to chromosome 16 and chromosome 4. Number of uniquely aligned reads (y axis, blue line) sequenced with PacBio that mapped to PKD1 and PKD2 . Off‐target amplification is discriminated from the main PKD1 gene sequences showing alignments to pseudogene homologous sequences at proximal loci (e.g., PKD1P1 , PKD1P5 , PKD1P6 ) (blue boxes). B: Mapping quality (MQ; in Phred quality scores; values > 90 were scaled down for visualization purposes), and sequencing depth (DP; in number of reads) of uniquely aligned molecules to PKD1 (NM_001009944.2) for the five LR‐PCR fragments amplified. Mapping quality of alignments with even coverage distribution along the amplified fragments (fragments), including regions with SDs, repetitive elements (repeats), and high GC content (GC%). Despite fragments A and E showing lower coverage, compared with the average sequencing depth of ≥421× (minimum ≥19×; maximum 1,528×), they had sufficient coverage for variant calling within the exon regions, including the first exons of PKD1 , with average coverage of ≥55× (minimum ≥24×; maximum 91×) (Supp. Table S4). C : We detected 1,506 intron variants (blue) and 177 coding or splice‐site variants (yellow). The predicted transcript effects of coding and splice‐site variants were quantified (bar chart) as log10 count (x axis)

Techniques Used: Sequencing, Variant Assay, Polymerase Chain Reaction, Amplification

12) Product Images from "Pseudoautosomal Region 1 Length Polymorphism in the Human Population"

Article Title: Pseudoautosomal Region 1 Length Polymorphism in the Human Population

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004578

Sequencing to validate the insertion and demonstrate recurrence. (A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.
Figure Legend Snippet: Sequencing to validate the insertion and demonstrate recurrence. (A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.

Techniques Used: Sequencing, Amplification, Translocation Assay

13) Product Images from "Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue"

Article Title: Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue

Journal: Scientific Reports

doi: 10.1038/s41598-018-20623-7

Phasing of de novo variants with variants known to belong to either the wildtype (MCF-7) or mutant (MDA-MB-231) genotype, exemplified by four Integrative Genomics Viewer (IGV) screenshots. ( A ) In the 0.1% mutant sample, position 7029 (T, red) phases together with reads containing the wildtype (MCF-7) variant at position 6776 (C, blue) but not the mutant (MDA-MB-231) variants at positions 7028 (T, red) and 8506 (C, blue). ( B ) In the 10% mutant sample, position 10406 (A, green) phases together with reads containing the mutant (MDA-MB-231) variants at position 11719 (A, green) and 12084 (T, red) but not the wildtype (MCF-7) variant at position 9966 (A, green). Note that position 10406 is covered by two amplicons, and thus detected by two independent observations. ( C ) In the 10% mutant sample, position 13623 (T, red) phases together with reads containing the mutant (MDA-MB-231) variants at position 12705 (T, red), 13966 (G, orange), 14470 (C, blue), 14766 (T, red) and 15310 (C, blue) but not the wildtype (MCF-7) variants at position 13260 (C, blue) and 14319 (C, blue). Note that position 13623 is covered by two amplicons, and thus detected by two independent observations. ( D ) In the 0.1% mutant sample, position 15897 (A, green) phases together with reads containing the wildtype (MCF-7) variants at position 15380 (G, orange) and 16148 (T, red) but not the mutant (MDA-MB-231) variants at position 15310 (C, blue), 16093 (C, blue), 16184 (A, green), 16189 (C, blue), 16223 (T, red), 16265 (G, orange) and 16278 (T, red). Horizontal is the DNA sequence, vertical the individual reads, and alignments sorted by base. Note that the position in IGV does not correspond to the rCRS position due to the use of an extended reference for alignment (see Materials and Methods). INDELs
Figure Legend Snippet: Phasing of de novo variants with variants known to belong to either the wildtype (MCF-7) or mutant (MDA-MB-231) genotype, exemplified by four Integrative Genomics Viewer (IGV) screenshots. ( A ) In the 0.1% mutant sample, position 7029 (T, red) phases together with reads containing the wildtype (MCF-7) variant at position 6776 (C, blue) but not the mutant (MDA-MB-231) variants at positions 7028 (T, red) and 8506 (C, blue). ( B ) In the 10% mutant sample, position 10406 (A, green) phases together with reads containing the mutant (MDA-MB-231) variants at position 11719 (A, green) and 12084 (T, red) but not the wildtype (MCF-7) variant at position 9966 (A, green). Note that position 10406 is covered by two amplicons, and thus detected by two independent observations. ( C ) In the 10% mutant sample, position 13623 (T, red) phases together with reads containing the mutant (MDA-MB-231) variants at position 12705 (T, red), 13966 (G, orange), 14470 (C, blue), 14766 (T, red) and 15310 (C, blue) but not the wildtype (MCF-7) variants at position 13260 (C, blue) and 14319 (C, blue). Note that position 13623 is covered by two amplicons, and thus detected by two independent observations. ( D ) In the 0.1% mutant sample, position 15897 (A, green) phases together with reads containing the wildtype (MCF-7) variants at position 15380 (G, orange) and 16148 (T, red) but not the mutant (MDA-MB-231) variants at position 15310 (C, blue), 16093 (C, blue), 16184 (A, green), 16189 (C, blue), 16223 (T, red), 16265 (G, orange) and 16278 (T, red). Horizontal is the DNA sequence, vertical the individual reads, and alignments sorted by base. Note that the position in IGV does not correspond to the rCRS position due to the use of an extended reference for alignment (see Materials and Methods). INDELs

Techniques Used: Mutagenesis, Multiple Displacement Amplification, Variant Assay, Sequencing

14) Product Images from "A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium"

Article Title: A High-Throughput Approach for Identification of Nontuberculous Mycobacteria in Drinking Water Reveals Relationship between Water Age and Mycobacterium avium

Journal: mBio

doi: 10.1128/mBio.02354-17

Schematic overview of the PacBio barcoding approach. For simplicity, this schematic shows how amplicon libraries are created from two separate samples. The approach includes two PCRs, the first specific to the target with M13 motifs on the 5′ and 3′ ends of the forward and reverse primers, respectively. The second PCR uses the product from step 1 and involves the addition of unique forward and reverse barcodes to the amplicons. Overall, the amplicon library created for each sample possesses a unique combination of barcodes, allowing both sample libraries to be pooled and distinguished postsequencing. Upon pooling, equal concentrations of the amplicon libraries (step 4) are submitted for PacBio sequencing by the CCS approach entailing circularization of the amplicons (SMRTbell).
Figure Legend Snippet: Schematic overview of the PacBio barcoding approach. For simplicity, this schematic shows how amplicon libraries are created from two separate samples. The approach includes two PCRs, the first specific to the target with M13 motifs on the 5′ and 3′ ends of the forward and reverse primers, respectively. The second PCR uses the product from step 1 and involves the addition of unique forward and reverse barcodes to the amplicons. Overall, the amplicon library created for each sample possesses a unique combination of barcodes, allowing both sample libraries to be pooled and distinguished postsequencing. Upon pooling, equal concentrations of the amplicon libraries (step 4) are submitted for PacBio sequencing by the CCS approach entailing circularization of the amplicons (SMRTbell).

Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing

15) Product Images from "Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion"

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

Journal: NPJ Genomic Medicine

doi: 10.1038/s41525-017-0042-3

Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )
Figure Legend Snippet: Deletion breakpoint identification. The chromosome 7p14.3 genomic region is illustrated with tracks for the proband chromosomal microarray (CMA) results, genomic PCR mapping amplicon locations (1–9; green: amplified; red: did not amplify), and copy number variants detected among healthy individuals in the Database of Genomic Variants (DGV; blue: duplication; red: deletion) ( a ). Unambiguous breakpoint mapping was performed by long-read single molecule real-time (SMRT) sequencing (PacBio) of long-range PCR products that amplified across the deleted interval in the proband ( b ). These SMRT sequencing data were also aligned to a modified human genome reference that excluded the identified 72.8 kb deletion (chr7:33130616–33203409) ( c ), confirming that there were no other sequence alterations at the breakpoint locations. The precise deletion breakpoints were subsequently confirmed in the proband and both carrier parents by Sanger sequencing of the long-range PCR amplicons ( d )

Techniques Used: Microarray, Polymerase Chain Reaction, Amplification, Sequencing, Modification

16) Product Images from "Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations"

Article Title: Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations

Journal: Scientific Reports

doi: 10.1038/s41598-018-22428-0

PCR amplification of the MUC1 -VNTR. Depiction of amplicons spanning the MUC1 -VNTR of families F1 to F7 separated by agarose gel electrophoresis (0.7% gel). Product sizes range between 2000 and 5000 bp. C, water control (see Table 1 and Fig. 4a ).
Figure Legend Snippet: PCR amplification of the MUC1 -VNTR. Depiction of amplicons spanning the MUC1 -VNTR of families F1 to F7 separated by agarose gel electrophoresis (0.7% gel). Product sizes range between 2000 and 5000 bp. C, water control (see Table 1 and Fig. 4a ).

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

17) Product Images from "Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics"

Article Title: Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics

Journal: Scientific Reports

doi: 10.1038/srep35790

Protocol for using IPCR to evaluate the horizontal gene transfer potential of ARGs in the environment. After sample collection (1) and total DNA extraction (2), the DNA is digested with restriction enzymes and resulting fragments are self-ligated into circular DNA molecules (3). DNA flanking the ARG is amplified with IPCR using ARG targeting primers (4). The amplicons are sequenced using long read sequencing with PacBio SMRT cell technology and the ARG associated MGEs are identified (5).
Figure Legend Snippet: Protocol for using IPCR to evaluate the horizontal gene transfer potential of ARGs in the environment. After sample collection (1) and total DNA extraction (2), the DNA is digested with restriction enzymes and resulting fragments are self-ligated into circular DNA molecules (3). DNA flanking the ARG is amplified with IPCR using ARG targeting primers (4). The amplicons are sequenced using long read sequencing with PacBio SMRT cell technology and the ARG associated MGEs are identified (5).

Techniques Used: DNA Extraction, Amplification, Sequencing

18) Product Images from "Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis"

Article Title: Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007761

Categories of sequencing reads inferred from the PacBio NGS sequencing. (A) Categories of sequencing reads deriving from the gapC amplicon, (B) i6-tubA amplicon and (C) i5-tubA amplicon, respectively. Number and percentage of particular types of reads are given. The distribution of introns in all groups of reads is depicted. Lines represent the exons while circles represent the introns: black circles: conventional introns; white: nonconventional ones (not to scale). Introns are labelled i1 –i6. In addition, introns that exhibit intermediate features are marked with an asterisk. Double asterisk indicates the introns to which the reverse primer was targeted, thus not considered in the analysis of amplicons (introns to which primers annealed were inevitably present in the amplification products).
Figure Legend Snippet: Categories of sequencing reads inferred from the PacBio NGS sequencing. (A) Categories of sequencing reads deriving from the gapC amplicon, (B) i6-tubA amplicon and (C) i5-tubA amplicon, respectively. Number and percentage of particular types of reads are given. The distribution of introns in all groups of reads is depicted. Lines represent the exons while circles represent the introns: black circles: conventional introns; white: nonconventional ones (not to scale). Introns are labelled i1 –i6. In addition, introns that exhibit intermediate features are marked with an asterisk. Double asterisk indicates the introns to which the reverse primer was targeted, thus not considered in the analysis of amplicons (introns to which primers annealed were inevitably present in the amplification products).

Techniques Used: Sequencing, Next-Generation Sequencing, Amplification

19) Product Images from "Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution"

Article Title: Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

Journal: bioRxiv

doi: 10.1101/2019.12.20.885061

Enzymatic deamination preserves the integrity of the DNA: a. qPCR results show the quantities of undamaged amplifiable DNA templates of different sizes after the enzymatic deamination (green) and bisulfite treatments (orange and blue). All quantifications are normalized to the values obtained for the enzymatic deamination experiments. b. Agilent 2100 Bioanalyzer trace on RNA 6000 pico chip comparing equal amounts of mouse E14 genomic DNA sheared to an average of 15 kb and treated with sodium bisulfite (green), BGT and APOBEC3A (red), or TET2 and APOBEC3A (blue) over the control ssDNA (magenta). Bisulfite treatment fragmented the DNA to an average of 800 bp, while enzymatically treated DNA show no notable size differences compared to control DNA. c. Agarose gel images of end-point PCR of six amplicons ranging from 388–4229 bp illustrating upper amplicon size limit for sodium bisulfite, TET2 and APOBEC3A, or BGT and APOBEC3A treated E14 genomic DNA. d. 731 bp amplicons from the agarose gels showed in (c) were cloned, sequenced and the methylation status determined for bisulfite (left panel), enzymatically converted for 5-mC (center panel) and 5-hmC (right panel) E14 genomic DNA. Open and closed circles indicate unmethylated and methylated, respectively.
Figure Legend Snippet: Enzymatic deamination preserves the integrity of the DNA: a. qPCR results show the quantities of undamaged amplifiable DNA templates of different sizes after the enzymatic deamination (green) and bisulfite treatments (orange and blue). All quantifications are normalized to the values obtained for the enzymatic deamination experiments. b. Agilent 2100 Bioanalyzer trace on RNA 6000 pico chip comparing equal amounts of mouse E14 genomic DNA sheared to an average of 15 kb and treated with sodium bisulfite (green), BGT and APOBEC3A (red), or TET2 and APOBEC3A (blue) over the control ssDNA (magenta). Bisulfite treatment fragmented the DNA to an average of 800 bp, while enzymatically treated DNA show no notable size differences compared to control DNA. c. Agarose gel images of end-point PCR of six amplicons ranging from 388–4229 bp illustrating upper amplicon size limit for sodium bisulfite, TET2 and APOBEC3A, or BGT and APOBEC3A treated E14 genomic DNA. d. 731 bp amplicons from the agarose gels showed in (c) were cloned, sequenced and the methylation status determined for bisulfite (left panel), enzymatically converted for 5-mC (center panel) and 5-hmC (right panel) E14 genomic DNA. Open and closed circles indicate unmethylated and methylated, respectively.

Techniques Used: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Clone Assay, Methylation

20) 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

Related Articles

Sequencing:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: .. Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA). .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS.

Polymerase Chain Reaction:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: .. Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA). .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS.

Amplification:

Article Title: Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion
Article Snippet: .. Long-read SMRT sequencing : Long-range PCR amplification across the identified breakpoint regions was accomplished using primers targeted to unique DNA sequences flanking the approximated deletion coordinates, and these amplicons were subjected to SMRTbell library construction and long-read SMRT sequencing (Pacific Biosciences, Menlo Park, CA). .. Long-range PCR reactions were performed in 50 µl containing ~100 ng of DNA, 1× LA PCR buffer II (TaKaRa), 0.4 µM of barcoded forward and reverse primers (Supplemental Table ), 0.4 mM dNTPs, 1 µL DMSO, and 2.5 units of TaKaRa LA Taq HS.

Construct:

Article Title: Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea, et al. Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea
Article Snippet: .. SMRTbell libraries were constructed (each containing amplicons from nine samples) and sequenced on a PacBio RSII system using the P6‐C4 chemistry with a 6‐hr movie time (Pacific Biosciences). .. 2.3 16S rRNA sequence data analysis To obtain the circular consensus sequences (CCS) with a minimum of five full passes, PacBio raw reads were demultiplexed and processed using the RS‐ReadsOfInsert protocol (SMRT Analysis software version 2.3).

Article Title: Dynamics of coral‐associated microbiomes during a thermal bleaching event, et al. Dynamics of coral‐associated microbiomes during a thermal bleaching event
Article Snippet: .. A total of two SMRTbell libraries were constructed (each containing amplicons from 9 to 10 samples) and sequenced on a PacBio RSII system, using the P6‐C4 polymerase and chemistry with a 360‐min movie time. .. 2.3 16S rRNA and ITS sequence data analysis RS‐ReadsOfInsert protocol (SMRT Analysis software version 2.3) was used to demultiplex and process PacBio raw reads to obtain consensus sequences with a minimum of five full passes.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis
Article Snippet: .. The amplicons obtained as a result of RT-PCR reactions for the gapC and tubA genes were sequenced commercially in the Museum and Institute of Zoology of the Polish Academy of Science, Warsaw, Poland, on the PacBio RS II (Pacific Biosciences) instrument. ..

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    Pacific Biosciences pacbio amplicon sequencing
    Sequencing to validate the insertion and demonstrate recurrence. (A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the <t>amplicon.</t> (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through <t>PacBio</t> amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.
    Pacbio Amplicon Sequencing, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pacific Biosciences amplicon sequence analysis pacbio smrt sequence data
    Graphical representation of the C3S-LAA process and pipeline. a Raw reads comprised of multiple subreads are depicted for three different amplicons [green, fuchsia and blue boxes; different shades of color are used to portray variable subread sequence qualities (darker shading portrays higher quality)]. Subreads are separated by a shared adapter sequence (grey boxes). The higher quality CCS read for each raw read is used to cluster the corresponding raw reads into CCS-based cluster groups. Error correction is performed per CCS-based cluster, producing top quality consequences sequences, followed by assembly of any overlapping consensus sequences. b A single run parameters file is used by all components of the pipeline. The grey highlighted rectangles represent two main steps of C3S-LAA. (i) Using the CCS reads generated by the <t>SMRT</t> analysis reads of insert protocol, C3S clusters the raw reads according to each barcode-primer pair combination, producing files of read identifiers to whitelist the corresponding raw reads. (ii) Raw read clusters are passed to Quiver to generate <t>amplicon-specific</t> consensus sequences, which are then passed to Minimus for sequence assembly. Rectangles with folded corners represent single files or multiple files (depicted as stacks of files) and those with rounded edges represent scripts and tools. Arrows indicates output files that are generated. Connecting lines with dots at one end depict input files, with the dot corresponding to the source data for the connected script or tool
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    Pacific Biosciences multiplex amplicons
    A histogram showing CCS read length distribution of 16S rRNA <t>amplicons</t>
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    Pacific Biosciences amplicon libraries
    Sequence alignment highlighting a recurring insertion error in some bootstrap samples. The alignment corresponds to the consensus sequence for a part of the <t>amplicon</t> from a locus_6_7045710_7052049 (Query) and b locus_1_25390617_25396540 (Query) on maize chromosome 6 and 1 respectively compared to the B73 v3 reference sequence (Sbjct)
    Amplicon Libraries, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Sequencing to validate the insertion and demonstrate recurrence. (A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.

    Journal: PLoS Genetics

    Article Title: Pseudoautosomal Region 1 Length Polymorphism in the Human Population

    doi: 10.1371/journal.pgen.1004578

    Figure Lengend Snippet: Sequencing to validate the insertion and demonstrate recurrence. (A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.

    Article Snippet: PacBio amplicon sequencing of the duplicon border identified variants upstream of the Sanger sequencing for rs211654, rs10625422, and rs211655 in all samples with longer amplicon lengths ( , all samples except P10-13).

    Techniques: Sequencing, Amplification, Translocation Assay

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

    Journal: BMC Bioinformatics

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

    doi: 10.1186/s12859-018-2293-0

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

    Article Snippet: Improving the accuracy of amplicon sequence analysis PacBio SMRT sequence data from a pooled library of long-range PCR amplicons was previously produced and used for part of this study [ ].

    Techniques: Sequencing, Generated, Amplification

    A histogram showing CCS read length distribution of 16S rRNA amplicons

    Journal: MicrobiologyOpen

    Article Title: Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea, et al. Heat‐induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea

    doi: 10.1002/mbo3.935

    Figure Lengend Snippet: A histogram showing CCS read length distribution of 16S rRNA amplicons

    Article Snippet: To multiplex amplicons from nine samples into one SMRTbell library, we used 27F and 1492R primers tailed with 16‐base PacBio barcodes at their 5’ ends ( ://github.com/PacificBiosciences/Bioinformatics-Training/blob/master/barcoding/pacbio_384_barcodes.fasta ).

    Techniques:

    Sequence alignment highlighting a recurring insertion error in some bootstrap samples. The alignment corresponds to the consensus sequence for a part of the amplicon from a locus_6_7045710_7052049 (Query) and b locus_1_25390617_25396540 (Query) on maize chromosome 6 and 1 respectively compared to the B73 v3 reference sequence (Sbjct)

    Journal: BMC Bioinformatics

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

    doi: 10.1186/s12859-018-2293-0

    Figure Lengend Snippet: Sequence alignment highlighting a recurring insertion error in some bootstrap samples. The alignment corresponds to the consensus sequence for a part of the amplicon from a locus_6_7045710_7052049 (Query) and b locus_1_25390617_25396540 (Query) on maize chromosome 6 and 1 respectively compared to the B73 v3 reference sequence (Sbjct)

    Article Snippet: It may be possible to use a less stringent clustering algorithm, however, the fragment lengths of most amplicon libraries are expected to be well below the current and increasingly long read lengths of PacBio data, such that highly accurate CCS reads would be available for clustering.

    Techniques: Sequencing, Amplification

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

    Journal: BMC Bioinformatics

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

    doi: 10.1186/s12859-018-2293-0

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

    Article Snippet: It may be possible to use a less stringent clustering algorithm, however, the fragment lengths of most amplicon libraries are expected to be well below the current and increasingly long read lengths of PacBio data, such that highly accurate CCS reads would be available for clustering.

    Techniques: Sequencing, Generated, Amplification