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    Service Plan for Ion Torrent Next Generation Sequencing System
    Description:
    To protect your Ion Torrent next generation sequencing NGS system the best time to plan for service is at the time of instrument purchase A proactive upfront service plan provides • Greater discounts for bundling your instrument and service together • Multi year plans that provide immunity to annual price increases • Service that is easier to budget for than “breakdown service repairs For the level of support that best meets your laboratory s needs and budget choose from flexible service plans and a variety of value added services for your instrument AB Assurance Service Plan The AB Assurance Service Plan is ideal for laboratories that heavily utilize their instruments and helps to reduce the risk of downtime by providing proactive maintenance service Should your instrument require a repair the AB Assurance coverage includes parts labor and travel at no additional cost Additionally with this plan receive • On site guaranteed 2 day response time • Scheduled on site planned maintenance PM • Remote diagnostics • Computer repair and replacement • Priority access to Remote Service Engineer Certain exclusions apply Contact your Services and Support Representative for details at service sales thermofisher com Availability limited in some geographic areas
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    ZG11SCIONCHEF
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    Instruments and Equipment
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    Ion Torrent™ Next-Generation Sequencing|Service & Support Plans for Ion Torrent™ Next-Generation Sequencing|Sequencing
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    Structured Review

    Thermo Fisher ngs sequencing kits
    The mean nucleotide percentage, N reads and average gap per sequencing method. The graph indicates a similarity in the nucleotide consistency between the sequencing methods. The SP method has an 8.9 and <t>SISPA</t> has a 10.3 times higher N reads compared to the <t>NGS</t> sequencer kit average. The Proton torrent had the least N reads, approximately 10 times lower than the other NGS sequencing kits. All the sequencing kits (Illumina, IonTorrrent and Proton Torrent) had the lowest N frequency.
    To protect your Ion Torrent next generation sequencing NGS system the best time to plan for service is at the time of instrument purchase A proactive upfront service plan provides • Greater discounts for bundling your instrument and service together • Multi year plans that provide immunity to annual price increases • Service that is easier to budget for than “breakdown service repairs For the level of support that best meets your laboratory s needs and budget choose from flexible service plans and a variety of value added services for your instrument AB Assurance Service Plan The AB Assurance Service Plan is ideal for laboratories that heavily utilize their instruments and helps to reduce the risk of downtime by providing proactive maintenance service Should your instrument require a repair the AB Assurance coverage includes parts labor and travel at no additional cost Additionally with this plan receive • On site guaranteed 2 day response time • Scheduled on site planned maintenance PM • Remote diagnostics • Computer repair and replacement • Priority access to Remote Service Engineer Certain exclusions apply Contact your Services and Support Representative for details at service sales thermofisher com Availability limited in some geographic areas
    https://www.bioz.com/result/ngs sequencing kits/product/Thermo Fisher
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    Images

    1) Product Images from "Evaluating methods for Avian avulavirus-1 whole genome sequencing"

    Article Title: Evaluating methods for Avian avulavirus-1 whole genome sequencing

    Journal: Gene: X

    doi: 10.1016/j.gene.2019.100004

    The mean nucleotide percentage, N reads and average gap per sequencing method. The graph indicates a similarity in the nucleotide consistency between the sequencing methods. The SP method has an 8.9 and SISPA has a 10.3 times higher N reads compared to the NGS sequencer kit average. The Proton torrent had the least N reads, approximately 10 times lower than the other NGS sequencing kits. All the sequencing kits (Illumina, IonTorrrent and Proton Torrent) had the lowest N frequency.
    Figure Legend Snippet: The mean nucleotide percentage, N reads and average gap per sequencing method. The graph indicates a similarity in the nucleotide consistency between the sequencing methods. The SP method has an 8.9 and SISPA has a 10.3 times higher N reads compared to the NGS sequencer kit average. The Proton torrent had the least N reads, approximately 10 times lower than the other NGS sequencing kits. All the sequencing kits (Illumina, IonTorrrent and Proton Torrent) had the lowest N frequency.

    Techniques Used: Sequencing, Next-Generation Sequencing

    Average coverage of each position in the AAvV-1 viral genome sequences, per method. The viruses were sequenced using three different methods including the specific primers (34), SISPA (57) and 96 samples via NGS sequencing kits (8 IonTorrent, 84 Proton Torrent, 4 Illumina). In all the methods a drop in the coverage near nucleotide 1700 is seen, possibly due to the G-quadruplexes. The Illumina and Proton Torrent have the highest coverage. The lowest coverage was obtained by the specific primers and SISPA methods (sequenced via the Ion Torrent).
    Figure Legend Snippet: Average coverage of each position in the AAvV-1 viral genome sequences, per method. The viruses were sequenced using three different methods including the specific primers (34), SISPA (57) and 96 samples via NGS sequencing kits (8 IonTorrent, 84 Proton Torrent, 4 Illumina). In all the methods a drop in the coverage near nucleotide 1700 is seen, possibly due to the G-quadruplexes. The Illumina and Proton Torrent have the highest coverage. The lowest coverage was obtained by the specific primers and SISPA methods (sequenced via the Ion Torrent).

    Techniques Used: Next-Generation Sequencing, Sequencing

    Related Articles

    Variant Assay:

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing
    Article Snippet: .. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. .. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.

    Next-Generation Sequencing:

    Article Title: Currently Applied Molecular Assays for Identifying ESR1 Mutations in Patients with Advanced Breast Cancer
    Article Snippet: Among our included studies, pyrosequencing was performed for sequencing ESR1 exon 5. .. In addition, it was also used to confirm all mutations in ESR1 identified using the Ion Torrent NGS platform [ ]. ..

    Article Title: The AURORA pilot study for molecular screening of patients with advanced breast cancer–a study of the breast international group
    Article Snippet: Nonetheless, this approach was applied to each copy number platform for 14 (54%) patients for whom all three data types were available and the segmented Log2 ratios were compared genome-wide using the Spearman’s correlation. .. The distribution of correlation coefficients comparing the SNP array to the NGS data was bimodal for the Ion Torrent platform with 5 (29%) samples displaying a poor correlation of ρ < 0.5 (Fig. ). .. The median correlation coefficients were ρ = 0.615 and ρ = 0.745 for the Ion Torrent and Illumina NGS platforms respectively.

    Article Title: NGS-based deep bisulfite sequencing
    Article Snippet: In Steps 2 and 3, this library of PCR products goes through the end repair reaction followed by size selection on a 2% agarose gel. .. In Step 4, the eluted DNA from the agarose gel is ligated to a set of duplex adaptors that are compatible with NGS platforms (in our case, Ion Torrent P1 and A adaptor) ( B and C). .. During this step, multiplexing can be achieved with a set of A adaptors with different barcodes ( ).

    Polymerase Chain Reaction:

    Article Title: LINE-1 retrotransposition impacts the genome of human pre-implantation embryos and extraembryonic tissues
    Article Snippet: Briefly, WGA-gDNAs were fragmented into 1kb fragments by sonication, end-repaired and linker-ligated. .. Separate suppression PCR reactions amplify L1Hs-Ta 5’- and 3’-junctions and add sample-specific barcodes, as well as Ion Torrent sequencing adapters. .. Libraries were multiplexed two-by-two and each pool was sequenced using a 318 Chip with a 400bp sequencing kit.

    Sequencing:

    Article Title: LINE-1 retrotransposition impacts the genome of human pre-implantation embryos and extraembryonic tissues
    Article Snippet: Briefly, WGA-gDNAs were fragmented into 1kb fragments by sonication, end-repaired and linker-ligated. .. Separate suppression PCR reactions amplify L1Hs-Ta 5’- and 3’-junctions and add sample-specific barcodes, as well as Ion Torrent sequencing adapters. .. Libraries were multiplexed two-by-two and each pool was sequenced using a 318 Chip with a 400bp sequencing kit.

    Article Title: High-Throughput Identification of Adapters in Single-Read Sequencing Data
    Article Snippet: Additional file 3 : Information on SOLiD sequencing datasets. .. Additional file 4 : Information on Ion Torrent sequencing datasets. .. Additional file 5 : adapt_find results for Illumina sequencing datasets.

    Agarose Gel Electrophoresis:

    Article Title: NGS-based deep bisulfite sequencing
    Article Snippet: In Steps 2 and 3, this library of PCR products goes through the end repair reaction followed by size selection on a 2% agarose gel. .. In Step 4, the eluted DNA from the agarose gel is ligated to a set of duplex adaptors that are compatible with NGS platforms (in our case, Ion Torrent P1 and A adaptor) ( B and C). .. During this step, multiplexing can be achieved with a set of A adaptors with different barcodes ( ).

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    Thermo Fisher cd4 t cell negative
    Design and Validation of Treg-specific Usp22 Knockout Mice. a) Diagram of the murine  Usp22  locus. Targeting vector contains IRES-lacZ and a neo cassette inserted into exon 2. b) Genotyping by PCR showed a 600-bp band for the wild-type allele and a 400-bp band for mutant allele, simultaneously in the homozygous floxed (f/f) mice. c) Western blot analysis of Usp22 in CD4 + CD25 -  conventional T cells (Tconv) and CD4 + CD25 +  Treg cells isolated from WT and KO mice. Tubulin was used as a loading control. d) Statistical analysis of CD4 + CD25 + Foxp3 +  Treg frequencies, n=4, corresponding to   Figure 2c . e) iTreg differentiations from WT and KO naïve CD4 +  T cells with titration of TGF-β.
    Cd4 T Cell Negative, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript double stranded cdna synthesis kit
    A schematic diagram of the procedure for expression tag library preparation. Double-stranded <t>cDNA</t> was synthesized from mRNA using a <t>biotinylated</t> adaptor oligo(dT) primer. After a digestion with Nla III, the 3′ end cDNA fragments anchored to streptavidin magnetic beads were ligated to the adaptor 1_GT, the adaptor 1_CT, the adaptor 1_AC, and the adaptor 1_TC for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively. The adaptor 1-ligated cDNA fragments were digested with Eco P15I, and the released fragments were ligated to the adaptor 2. The adaptor 1-tag–adator 2 fragments were amplified using the GEX PCR primer set. The amplified fragments (∼104 bp) were separated by PAGE. Four independent library solutions (Cd0, Cd3h, Cd1d, and Cd3d) were mixed in an equal amount of DNA, and the blended library solution was subjected to high-throughput sequencing using an Illumina Genome Analyzer. Underlined characters indicate Nla III and Eco P15I recognition sites. Bold ‘XX’ indicates library coding nucleotides, and ‘GT’, ‘CT’, ‘AC’, and ‘TC’ were used for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively.
    Superscript Double Stranded Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher amplicon sequencing libraries
    Deletion alleles seen in the SULT-OR gene in <t>amplicon</t> sequencing reads from treated (A) and control (B) adults (left), sporocysts (centre), and eggs (right), showing alleles that contain a single internal deletion and no internal insertions with respect to the reference amplicon. The y-axis shows deletion alleles sorted by the number of reads supporting them, with the alleles supported by the most reads at the bottom. Alleles supported by > 500 reads in red, alleles supported by 101-500 reads in dark orange, alleles supported by 11-100 reads in pale orange, and alleles supported by 1-10 reads in pale green. The x-axis shows the position of the deletion along the reference amplicon, with a blue vertical line at the predicted Cas9 cut site.
    Amplicon Sequencing Libraries, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Design and Validation of Treg-specific Usp22 Knockout Mice. a) Diagram of the murine  Usp22  locus. Targeting vector contains IRES-lacZ and a neo cassette inserted into exon 2. b) Genotyping by PCR showed a 600-bp band for the wild-type allele and a 400-bp band for mutant allele, simultaneously in the homozygous floxed (f/f) mice. c) Western blot analysis of Usp22 in CD4 + CD25 -  conventional T cells (Tconv) and CD4 + CD25 +  Treg cells isolated from WT and KO mice. Tubulin was used as a loading control. d) Statistical analysis of CD4 + CD25 + Foxp3 +  Treg frequencies, n=4, corresponding to   Figure 2c . e) iTreg differentiations from WT and KO naïve CD4 +  T cells with titration of TGF-β.

    Journal: bioRxiv

    Article Title: CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

    doi: 10.1101/2020.02.26.966911

    Figure Lengend Snippet: Design and Validation of Treg-specific Usp22 Knockout Mice. a) Diagram of the murine Usp22 locus. Targeting vector contains IRES-lacZ and a neo cassette inserted into exon 2. b) Genotyping by PCR showed a 600-bp band for the wild-type allele and a 400-bp band for mutant allele, simultaneously in the homozygous floxed (f/f) mice. c) Western blot analysis of Usp22 in CD4 + CD25 - conventional T cells (Tconv) and CD4 + CD25 + Treg cells isolated from WT and KO mice. Tubulin was used as a loading control. d) Statistical analysis of CD4 + CD25 + Foxp3 + Treg frequencies, n=4, corresponding to Figure 2c . e) iTreg differentiations from WT and KO naïve CD4 + T cells with titration of TGF-β.

    Article Snippet: Cell Isolation and Flow Cytometry for Analysis of Usp22 KO Mice Peripheral T cells were isolated from mouse spleen by a CD4+ T-cell negative (Stem Cell) or positive selection kit (Invitrogen).

    Techniques: Knock-Out, Mouse Assay, Plasmid Preparation, Polymerase Chain Reaction, Mutagenesis, Western Blot, Isolation, Titration

    Design and Quality Control of Targeted Pooled CRISPR Screen in Primary Mouse Tregs a) Design strategy for selection of genes for unbiased targeted library of 493 targets, including 490 nuclear factors and 3 control targets (NT, GFP, and RFP). Genes were selected based on gene ontology (GO) annotation and then sub-selected based on highest expression across any CD4 T cell subset for a total of 2,000 sgRNAs. b) Diagram of MSCV expression vector with Thy1.1 reporter used for retroviral transduction of the sgRNA library. c) Detailed timeline schematic of the 12-day targeted screen pipeline. Arrows indicate when the cells were split and media was replenished. d) Retroviral transduction efficiency of the targeted library in primary mouse Tregs shown by Thy1.1 surface expression measured by flow cytometry. The infection was scaled to achieve a high efficiency multiplicity of infection. e) Foxp3 expression from screen input, output and control cells measured by flow cytometry. Top: Foxp3 expression from input Foxp3 + purified Tregs as measured by GFP expression on Day 0. Middle: Foxp3 expression as measured by endogenous intracellular staining from control Tregs (not transduced with library) on Day 12. Bottom: Foxp3 expression as measured by endogenous intracellular staining from screen Tregs (transduced with library) on Day 12. f) Targeted screen (2,000 guides) shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3 low cells (blue). Non-targeting control (NT Ctrl) sgRNAs were evenly distributed across the cell populations (black). g) Distribution of read counts after next generation sequencing of sgRNAs of sorted cell populations, Foxp3 high and Foxp3 low .

    Journal: bioRxiv

    Article Title: CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

    doi: 10.1101/2020.02.26.966911

    Figure Lengend Snippet: Design and Quality Control of Targeted Pooled CRISPR Screen in Primary Mouse Tregs a) Design strategy for selection of genes for unbiased targeted library of 493 targets, including 490 nuclear factors and 3 control targets (NT, GFP, and RFP). Genes were selected based on gene ontology (GO) annotation and then sub-selected based on highest expression across any CD4 T cell subset for a total of 2,000 sgRNAs. b) Diagram of MSCV expression vector with Thy1.1 reporter used for retroviral transduction of the sgRNA library. c) Detailed timeline schematic of the 12-day targeted screen pipeline. Arrows indicate when the cells were split and media was replenished. d) Retroviral transduction efficiency of the targeted library in primary mouse Tregs shown by Thy1.1 surface expression measured by flow cytometry. The infection was scaled to achieve a high efficiency multiplicity of infection. e) Foxp3 expression from screen input, output and control cells measured by flow cytometry. Top: Foxp3 expression from input Foxp3 + purified Tregs as measured by GFP expression on Day 0. Middle: Foxp3 expression as measured by endogenous intracellular staining from control Tregs (not transduced with library) on Day 12. Bottom: Foxp3 expression as measured by endogenous intracellular staining from screen Tregs (transduced with library) on Day 12. f) Targeted screen (2,000 guides) shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3 low cells (blue). Non-targeting control (NT Ctrl) sgRNAs were evenly distributed across the cell populations (black). g) Distribution of read counts after next generation sequencing of sgRNAs of sorted cell populations, Foxp3 high and Foxp3 low .

    Article Snippet: Cell Isolation and Flow Cytometry for Analysis of Usp22 KO Mice Peripheral T cells were isolated from mouse spleen by a CD4+ T-cell negative (Stem Cell) or positive selection kit (Invitrogen).

    Techniques: CRISPR, Selection, Expressing, Plasmid Preparation, Transduction, Flow Cytometry, Infection, Purification, Staining, Next-Generation Sequencing

    A schematic diagram of the procedure for expression tag library preparation. Double-stranded cDNA was synthesized from mRNA using a biotinylated adaptor oligo(dT) primer. After a digestion with Nla III, the 3′ end cDNA fragments anchored to streptavidin magnetic beads were ligated to the adaptor 1_GT, the adaptor 1_CT, the adaptor 1_AC, and the adaptor 1_TC for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively. The adaptor 1-ligated cDNA fragments were digested with Eco P15I, and the released fragments were ligated to the adaptor 2. The adaptor 1-tag–adator 2 fragments were amplified using the GEX PCR primer set. The amplified fragments (∼104 bp) were separated by PAGE. Four independent library solutions (Cd0, Cd3h, Cd1d, and Cd3d) were mixed in an equal amount of DNA, and the blended library solution was subjected to high-throughput sequencing using an Illumina Genome Analyzer. Underlined characters indicate Nla III and Eco P15I recognition sites. Bold ‘XX’ indicates library coding nucleotides, and ‘GT’, ‘CT’, ‘AC’, and ‘TC’ were used for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively.

    Journal: Journal of Experimental Botany

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum

    doi: 10.1093/jxb/erp313

    Figure Lengend Snippet: A schematic diagram of the procedure for expression tag library preparation. Double-stranded cDNA was synthesized from mRNA using a biotinylated adaptor oligo(dT) primer. After a digestion with Nla III, the 3′ end cDNA fragments anchored to streptavidin magnetic beads were ligated to the adaptor 1_GT, the adaptor 1_CT, the adaptor 1_AC, and the adaptor 1_TC for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively. The adaptor 1-ligated cDNA fragments were digested with Eco P15I, and the released fragments were ligated to the adaptor 2. The adaptor 1-tag–adator 2 fragments were amplified using the GEX PCR primer set. The amplified fragments (∼104 bp) were separated by PAGE. Four independent library solutions (Cd0, Cd3h, Cd1d, and Cd3d) were mixed in an equal amount of DNA, and the blended library solution was subjected to high-throughput sequencing using an Illumina Genome Analyzer. Underlined characters indicate Nla III and Eco P15I recognition sites. Bold ‘XX’ indicates library coding nucleotides, and ‘GT’, ‘CT’, ‘AC’, and ‘TC’ were used for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively.

    Article Snippet: Double-stranded cDNA was synthesized using the SuperScript Double Stranded cDNA Synthesis Kit (Invitrogen) and a biotinylated oligo(dT) primer (QIAGEN, Valencia, CA, USA).

    Techniques: Expressing, Synthesized, Magnetic Beads, Amplification, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing

    Deletion alleles seen in the SULT-OR gene in amplicon sequencing reads from treated (A) and control (B) adults (left), sporocysts (centre), and eggs (right), showing alleles that contain a single internal deletion and no internal insertions with respect to the reference amplicon. The y-axis shows deletion alleles sorted by the number of reads supporting them, with the alleles supported by the most reads at the bottom. Alleles supported by > 500 reads in red, alleles supported by 101-500 reads in dark orange, alleles supported by 11-100 reads in pale orange, and alleles supported by 1-10 reads in pale green. The x-axis shows the position of the deletion along the reference amplicon, with a blue vertical line at the predicted Cas9 cut site.

    Journal: bioRxiv

    Article Title: Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

    doi: 10.1101/2020.05.03.074831

    Figure Lengend Snippet: Deletion alleles seen in the SULT-OR gene in amplicon sequencing reads from treated (A) and control (B) adults (left), sporocysts (centre), and eggs (right), showing alleles that contain a single internal deletion and no internal insertions with respect to the reference amplicon. The y-axis shows deletion alleles sorted by the number of reads supporting them, with the alleles supported by the most reads at the bottom. Alleles supported by > 500 reads in red, alleles supported by 101-500 reads in dark orange, alleles supported by 11-100 reads in pale orange, and alleles supported by 1-10 reads in pale green. The x-axis shows the position of the deletion along the reference amplicon, with a blue vertical line at the predicted Cas9 cut site.

    Article Snippet: 2.5 DNA isolation and amplicon sequencing libraries A conventional phenol:chloroform:isoamyl alcohol (25:24:1) protocol was employed to isolate DNA from RNP-transfected parasites and all control groups.

    Techniques: Amplification, Sequencing

    (A) Frequency of deletions in NGS sequencing data, identified with the assistance of CRISPResso in three biological replicates from adults, two from sporocysts, and three from eggs. (B) CRISPR-induced deletions in adult worms and sporocysts. The positions of deletions found by CRISPResso in the reference amplicon are indicated, in three biological replicates of CRISPR-Cas9-treated adult samples (blue lines: experiment 1, tag 5; experiment 7, tag 50; experiment 11, tag 64) and matched adult control samples (red lines: experiment 1, tag 9; experiment 7, tag 82; experiment 11, tag 21), and in two biological replicates of CRISPR-Cas9-treated sporocysts (green lines: experiment 2, tag 6; experiment 11, tag 19) and matched sporocyst controls (orange lines: experiment 2, tag 15; experiment 11, tag 43). The black arrow shows the predicted Cas9 cut site. (C) Multi-sequence alignment of SULT-OR alleles with deletions found in CRISPR-Cas9-treated adult worms that are supported by > =50 reads and span the DSB site indicated with a red line, based on one of the treated adult replicates (experiment 1, tag 5). The common 34-bp deletion is highlighted in pale pink.

    Journal: bioRxiv

    Article Title: Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

    doi: 10.1101/2020.05.03.074831

    Figure Lengend Snippet: (A) Frequency of deletions in NGS sequencing data, identified with the assistance of CRISPResso in three biological replicates from adults, two from sporocysts, and three from eggs. (B) CRISPR-induced deletions in adult worms and sporocysts. The positions of deletions found by CRISPResso in the reference amplicon are indicated, in three biological replicates of CRISPR-Cas9-treated adult samples (blue lines: experiment 1, tag 5; experiment 7, tag 50; experiment 11, tag 64) and matched adult control samples (red lines: experiment 1, tag 9; experiment 7, tag 82; experiment 11, tag 21), and in two biological replicates of CRISPR-Cas9-treated sporocysts (green lines: experiment 2, tag 6; experiment 11, tag 19) and matched sporocyst controls (orange lines: experiment 2, tag 15; experiment 11, tag 43). The black arrow shows the predicted Cas9 cut site. (C) Multi-sequence alignment of SULT-OR alleles with deletions found in CRISPR-Cas9-treated adult worms that are supported by > =50 reads and span the DSB site indicated with a red line, based on one of the treated adult replicates (experiment 1, tag 5). The common 34-bp deletion is highlighted in pale pink.

    Article Snippet: 2.5 DNA isolation and amplicon sequencing libraries A conventional phenol:chloroform:isoamyl alcohol (25:24:1) protocol was employed to isolate DNA from RNP-transfected parasites and all control groups.

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

    (A) Gene model of SULT-OR ( Smp_089320 ), indicating the position of the two exons, one intron, and UTRs, spanning 4837 bp on the reverse strand of chromosome 6. Boxes filled in dark red represent the protein coding sequence. Schistosoma mansoni (PRJEA36577). Assembly: Smansoni_v7 (GCA_000237925.3). Region: Scaffold SM_V7_6:3,183,084-3,188,924. Adapted from WormBase ParaSite 14 [ 34 ]. (B) Nucleotide sequence of the start of exon 1 indicating location and sequence of gRNA target site, predicted double-stranded break (DSB), protospacer adjacent motif (PAM), and the PvuII restriction site. (C) Reference PCR amplicon, showing the positions of the gRNA, PAM, DSB, forward and reverse PCR primers, and forward and reverse sequence reads, as well as a SNP site found in many sequences reads. The diagrams are drawn to scale.

    Journal: bioRxiv

    Article Title: Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

    doi: 10.1101/2020.05.03.074831

    Figure Lengend Snippet: (A) Gene model of SULT-OR ( Smp_089320 ), indicating the position of the two exons, one intron, and UTRs, spanning 4837 bp on the reverse strand of chromosome 6. Boxes filled in dark red represent the protein coding sequence. Schistosoma mansoni (PRJEA36577). Assembly: Smansoni_v7 (GCA_000237925.3). Region: Scaffold SM_V7_6:3,183,084-3,188,924. Adapted from WormBase ParaSite 14 [ 34 ]. (B) Nucleotide sequence of the start of exon 1 indicating location and sequence of gRNA target site, predicted double-stranded break (DSB), protospacer adjacent motif (PAM), and the PvuII restriction site. (C) Reference PCR amplicon, showing the positions of the gRNA, PAM, DSB, forward and reverse PCR primers, and forward and reverse sequence reads, as well as a SNP site found in many sequences reads. The diagrams are drawn to scale.

    Article Snippet: 2.5 DNA isolation and amplicon sequencing libraries A conventional phenol:chloroform:isoamyl alcohol (25:24:1) protocol was employed to isolate DNA from RNP-transfected parasites and all control groups.

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification