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  • The Agilent Technologies

    SureSelect Platform for Target Enrichment

    Focus your next-gen sequencing on DNA that matters

    Kimberly Troutman

    Field Applications Scientist

    January 27th, 2011

  • Agenda

    Introduction: SureSelectTM

    2 Exome Approach for Genetic Diseases

    1

    3 Complex Diseases

    Custom Biomarker Discovery and Profiling4

    7 New SureSelect Products

    5 Targeted RNA Sequencing

    6 Kinome Kit

    Page 2

  • Page 3

    Target Enrichment: A Highly Enabling Process

    What?

    Also referred to as genome partitioning, targeted re-sequencing, DNA capture

    Captures genomic material of interest for next generation sequencer (i.e. Illumina, SOLiD, 454 etc)

    Why?

    Sequence your regions of interest!

    Enables focus on a subset of the genome

    Saves both time and money for downstream sequencing

    Identify homozygous and heterozygous

    variants in targets relative to the reference

    genome

    gDNA

    Enriched

    library

  • Page 4Page 4

    Agilents SureSelect Platform: Two Options

    SureSelect Target

    Enrichment System*

    Developed in collaboration

    with the Broad Institute

    Dr. Chad Nusbaum et al.

    SureSelect

    DNA Capture Array

    Developed in collaboration

    with Cold Spring Harbor

    Dr. Greg Hannon et al.

    *Flagship Method Released February 2009

    Agilent 60-mer Array

    244k & 1M features

    3 g gDNA

    1-5 g gDNA

    (with WGA)

    or

    20 g gDNA (unamplified)

    Released July 2009

  • Illumina GAIIx

    Illumina HiSeq

    SOLiD 3

    SOLiD 4

    5500

    GS FLX &

    GS JR

    Baits

    cRNA probes Long (120 bases) Biotin labeled

  • SureSelect Target Enrichment Kit Choices

    Product Target amount (Mb) Reactions/kit Product Definition

    Human X-

    demo3.05 5 Human X-chr Exons

    Human All

    Exon v138 5-10,000

    Catalog content from CCDS

    2008 plus >1000 ncRNA

    Human All

    Exon Plus

    38-50 plus up to 6.8 of

    custom content5-10,000

    Add custom content to All

    Exon catalog content

    Human All

    Exon v244 5-10,000

    CCDS Sept. 2009

    Plus additional RefSeq

    Human All

    Exon 50Mb50 5-10,000

    GENCODE content

    Most comprehensive coverage

    Multiplexable

    Kinome 3.2 5-10,000 All kinases

    Indexed

    custom

    content

  • SureSelect Kits Multiplexing Capability

    Target Enrichment Size

    Ranges

    Illumina AB SOLiD

    GA HiSeq 2000 Octet Quadrant Flow Cell Full Run

  • XT

  • Agilent SureSelectXT Kits

    gDNA kit + Library Prep kit + SureSelect Reagents

    = SureSelectXT Kit

    SureSelectXT Kit Coupled with an optimized gDNA prep and library prep kit, allows the

    use one kit for the entire, sample-prep-to-sequencing target enrichment workflow

    Kit composition

    gDNA Isolation Lysis buffer and enzymes required for isolation

    Library prep Buffers, reagents, enzymes and indexes needed for prep

    SureSelect Target Enrichment Kit Hybridization buffers and baits

    All kits are available in the XT format- catalog kits and custom content

    SureSelectXT All Exome & SureSelectXT All Exome Plus

    SureSelectXT Human Kinome & SureSelectXT Human X Chromosome

    SureSelectXT Custom from < 200 Kb to > 6.8 Mb (up to 34 Mb in Spring 2011)

    Illumina GAIIx and HiSeq 2000 (Protocol v1.0 Nov 2010)

    SOLiD 3 / 4 and 5500 (Available soon)

  • SureSelectXT complete sample to sequencer

    solutions for your target enrichment needsGenomic

    DNA prep

    Library prep

    (GA, SOLiD)

    Bioanalyzer,

    qPCR quant

    Manual Procedure for small

    number of samples

    Sequencer

    Page 11

  • Agenda

    Introduction: SureSelectTM

    2 Exome Approach for Genetic Diseases

    1

    3 Complex Diseases

    Custom Biomarker Discovery and Profiling4

    7 New SureSelect Products

    5 Targeted RNA Sequencing

    6 Kinome Kit

    Page 12

  • Exon Capture is a Powerful Tool to Study

    Mendelian Diseases

    Mendelian diseases are caused by coding mutations (with some exceptions)

    Exons are only ~1-1.4 % of human genome (30-50Mb)

    Primarily protein coding regions

    Advantages:

    Much less sequencing

    ~5% of WGS, so up to 20x more samples

    Why coding?

    More interpretable

    Easier to follow up

    Especially adapted to study of Mendelian diseases

    CCDS exons v1 CCDS + RefSeq 38 Mb v2 (Broad)

    GENCODE 50 Mb (Sanger)

    Includes ncRNA

    All Exons on X chromosomes 7674 exons

    3 Mb

    Page 13

  • Applications to Mendelian disorders

    Page 16

    and many more to come

  • Page 17

    SureSelect Human All Exon Kits

    All Exon v1 All Exon v2 All Exon50 Mb

    CCDS Sept. 2008

    CCDS Sept. 2008

    + additional RefSeq

    content including

    CCDS Sept. 2009

    exons

    GENCODE and

    Sanger (includes

    CCDS and Broad

    defined v2 content as

    well)

    CCDS (Nov. 2010) 89.6% 98.2% 99.5%

    CNV (Mar. 2010) 23.98% 27.49% 30.62%

    Ensembl (Aug. 2010) 79.9% 90.9% 96.2%

    miRNA (miRBase 14) 90.0% 90.0% 92.8%

    GenBank (6/16/2010) 75.96% 89.07% 90.74%

    RefSeq Genes (Nov. 2010) 85.0% 96.9% 99.0%

    RefSeq Transcripts

    (6/16/2010)88.85% 95.07% 97.50%

    Target Size 38Mb 44Mb 50Mb

    Developed with Broad Broad Sanger

    Human All Exon kits can be customized (PLUS) with up to 6.8 Mb additional custom content Human All Exon kits can be multiplexed on SOLiD4 and HiSeq2000

  • Human All Exon 50Mb 2x76 bp, 50-60M HQ Reads

    Page 18

    76.32%

    85.07%

    96.65%

    87.93%

    77.46%

    0%

    10%

    20%

    30%

    40%

    50%

    60%

    70%

    80%

    90%

    100%

    % on target +/-

    100bp

    Uniformity (3/4 mean with upper

    tail):

    % bases with 1x

    coverage

    % bases with 10x coverage

    % bases with 20x coverage

    The most comprehensive Human

    All Exon content available

    38 Mb design = a subset of 50 Mb

    Sequencing capacity:

    0.5-1 sample / lane GAIIx

    1-3 samples / lane HiSeq

    5-10 samples /full slide SOLiD4

    Chemistry recommended:

    PE 2x76 bp Illumina v4

    PE 50+25 SOLiD

    Multiplexing:

    Illumina

    SOLiD

  • Comparison of SNP Calls with HapMap

    Page 19

    99.1% 99.2%98.4% 98.0%

    95.7%94.9%

    70%

    75%

    80%

    85%

    90%

    95%

    100%

    Human All Exon v2 Human All Exon 50Mb

    GT is REF GT is variant HOM

    GT is variant HET

    99.8% 99.7%98.2% 98.1%98.5% 98.3%

    99.4% 99.3%

    70%

    75%

    80%

    85%

    90%

    95%

    100%

    Human All Exon v2 Human All Exon 50Mb

    GT is REF GT is variant HOM

    GT is variant HET OVERALL

    Genotype Concordance vs. HapMapGenotype Sensitivity vs. HapMap

  • All Exon Plus

    Page 20

    All Exon Library

    +

    Your Custom Library

    Enter Your Custom Regions in eArray

    CCDS exons

    >1000 ncRNAs

    38 Mb

    Your regions of

    interest (6.8 Mb)

    Is the Human All Exon Kit not hitting all of your regions

    of interest?

  • Human All Exon Plus Performance

    Page 21

    1 tube capture, 1 lane seq. at 2x76 bp on GAIIx = ~2 Gb

    0%

    10%

    20%

    30%

    40%

    50%

    60%

    70%

    80%

    90%

    100%

    Exome +

    0.87 Mb

    Exome +

    1.7 Mb

    Exome +

    3.4 Mb

    Exome +

    6.8 Mb

    Exome

    Control

    SNP Analysis vs. HapMap

    Sensitivity Concordance

    0

    5000

    10000

    15000

    20000

    25000

    30000

    35000

    Exome +

    0.87 Mb

    Exome +

    1.7 Mb

    Exome +

    3.4 Mb

    Exome +

    6.8 Mb

    Exome

    Control

    22394

    23224

    24337

    26352

    21976

    4480

    5528

    5816 6

    182

    5173

    No. S

    NP

    s

    SNP Analysis vs. dbSNP

    Concordant Novel Mismatched

  • Agenda

    Introduction: SureSelectTM

    2 Exome Approach for Genetic Diseases

    1

    3 Complex Diseases

    Custom Biomarker Discovery and Profiling4

    7 New SureSelect Products

    5 Targeted RNA Sequencing

    6 Kinome Kit

    Page 22

  • Beyond Mendelian Diseases: Complex diseases

    Page 23

  • Beyond Mendelian Diseases: Complex diseases

    Page 24

    25bp deletion

    7bp deletion

    10bp deletion

    11bp deletion

  • Agenda

    Introduction: SureSelectTM

    2 Exome Approach for Genetic Diseases

    1

    3 Complex Diseases

    Custom Biomarker Discovery and Profiling4

    7 New SureSelect Products

    5 Targeted RNA Sequencing

    6 Kinome Kit

    Page 25

  • Other Applications of Targeted Re-Sequencing

    Capture any custom genomic regions (introns, exons, UTRs, regulatory, etc.)

    Ideal for biomarkers discovery and profiling (e.g. cancer)

    Ideal for custom SNP follow-up

    Ideal for characterization of large sample cohorts

    Key enabling features:

    High throughput

    12 Illumina indexes / up to 96 samples per run

    16 SOLiD barcodes / up to 128 samples per run

    Only pay what you capture, scalable from 0.2 to 6.9 Mb (sweet spot for 3rd Gen

    Seq)

  • Page 27

    Target Enrichment Design Application in eArray

    eArray is a tool to d

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