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  • Cost-Effective Options using SureSelectXT SystemCost Effective Options using SureSelect System in Exome Sequencing and Targeted Applications

    XTXT

    Josh Wang, Ph.D.Field Application ScientistField Application ScientistFeb 8th, 2011

  • Cost-effective Options using SureSelectXT System

    Introduction to SureSelectXT Target Enrichment System

    SureSelectXT All Exon Kits and MultiplexingSureSelect All Exon Kits and Multiplexing

    SureSelectXT Targeted Kits and Multiplexing

    Custom capture kitsp

    Human Kinome kits (DNA capture & RNA capture)

    Bravo Automation for Library Preparation and Capture

  • SureSelectXT Target Enrichment SystemAddresses a major workflow bottleneck with a complete offering

    gDNA Extraction

    Library Prep

    j p g

  • SureSelectXT Target Enrichment System

    Illumina GAIIxIllumina HiSeq SOLiD 4 Roche 454SOLiD 5500XL

    Illumina ABI SOLiD Roche 454Illumina ABI SOLiD Roche 454Exome and Targeted SureSelect yes yes yesmultiplexing yes yes no

  • SureSelectXT Target Enrichment Kit ConfigurationsTarget amountProduct Target amount (catalog number) Product Definition

    Human All Exon v1 38 Mb CCDS Sept. 2008

    CCDS Sept 2009Human All Exon v2 44 Mb CCDS Sept. 2009 + additional RefSeq

    Human All Exon 50Mb 50 Mb GENCODE content most comprehensive coverage

    Human All Exon Plus 38-50 Mb + up to 6.8 Mb of custom contentadd custom content to All Exon catalog

    content

    Mouse All Exon 50 Mb mm9 build, Ensembl + RefSeq

    Kinome 3.2 Mb kinases and kinase related genes

    RNA capture Kinome 3.4 Mb transcripts for the kinome

  • Comparison: Target Enrichment System Applications

    Application Agilent SureSelectXT Brand N Brand I

    Exome Sequencing All Exon, All Exon v2, All Exon 50Mb, Exome v1, ExomeExome Sequencing All Exon Plus; mouse All Exon Exome v2 Exome

    GWAS follow-up custom SureSelectXT chip format onlyCancer Biomarker Discovery/Profiling Kinome DNA and RNA capture

    Targeted Kinome RNA capture, custom RNA gtranscriptome

    p ,SureSelect

    Clinical Research custom SureSelectXT (DNA, RNA)

    Microbial/viral seqdiscovery custom SureSelect

    XT

    All A il t S S l tXT kit i l ti b d d t ti f i dl All Agilent SureSelectXT kits are in-solution based and automation-friendly

  • SureSelect eArray Web for Target Enrichment Applications

  • Cost-effective Options using SureSelectXT System

    Introduction to SureSelectXT Target Enrichment System

    SureSelectXT All Exon Kits and MultiplexingSureSelect All Exon Kits and Multiplexing

    SureSelectXT Targeted Kits and Multiplexing

    Custom capture kitsp

    Human Kinome kits (DNA capture & RNA capture)

    Bravo Automation for Library Preparation and Capture

  • SureSelectTM Human All Exon Kit Comparison

    All Exon v1 All Exon v2 All Exon 50Mb

    CCDS Sept 2008

    CCDS Sept. 2008 + additional RefSeq content GENCODE and Sanger (includes CCDS and BroadCCDS Sept. 2008 qincluding CCDS Sept. 2009

    exons

    (includes CCDS and Broad defined v2 content as well)

    Target Size 38Mb 44Mb 50MbCCDS (Nov. 2010) 89.6% 98.2% 99.5%CCDS (Nov. 2010) 89.6% 98.2% 99.5%RefSeq Genes (Nov. 2010) 85.9% 96.9% 99.0%

    miRNA (miRBase v14) 90.0% 90.0% 92. 8%Ensembl (Aug 2010) 79 9% 90 9% 96 2%Ensembl (Aug. 2010) 79.9% 90.9% 96.2%GENCODE v4 80.9% 92.9% 97.0Addition of Custom Exome (

  • All Exon Kit Performance: Accuracy and Repeatability

    human all exon v12x76bp ~ 4Gb

    val(

    sam

    ple1

    )

    180160

    ~70% on-target~90% targeted bases at >10X~80% targeted bases at >20X

    2dep

    thpe

    rint

    erv 140

    120

    100

    80

    DNA Sample NA18507 NA10831Hom Het Hom Het

    T t l SNP 49 556 10 195 50 174 9 637 R2 = 0.956

    age

    Cov

    erag

    ed

    6040

    20

    0

    Total SNPs 49,556 10,195 50,174 9.637% Covered (> 1 read) 99.30 99.22 99.37 99.19

    % Called 95.33 81.90 96.00 83.36% Concordant

    Ave

    ra 00 20 40 60 80 100 120 140 160180

    Average Coverage depth per interval (sample2)

    % Concordant Calls 99.56 99.94 99.58 99.96

  • Human All Exon 50Mb 5Gb coverage

    Most comprehensive Human All Exon content available

    Sequencing capacity: 0.5-1 sample / lane GAIIx 1-3 samples / lane HiSeq 1-3 samples / lane HiSeq 5-10 samples /flowcell SOLiD4

    Chemistry recommended: PE 2x76bp Illumina PE 50+25 SOLiD

    Multiplexing:Multiplexing: Illumina SOLiD

    Page Page 1111 Agilent SureSelect PlatformEnabling Products for the

  • Comparison of SNP calls with HapMapGenotype Concordance vs HapMapG t S iti it H M

    99.1% 99.2%98.4% 98.0%

    95.7% 94 9%

    100%99.8% 99.7%

    98.2% 98.1%98.5% 98.3%99.4% 99.3%

    95%

    100%

    Genotype Concordance vs. HapMapGenotype Sensitivity vs. HapMap

    94.9%

    90%

    95%

    90%

    95%

    80%

    85%

    80%

    85%

    70%

    75%

    70%

    75%

    70%Human All Exon v2 Human All Exon

    50MbGT is REFGT is variant HOM

    Human All Exon v2 Human All Exon 50Mb

    GT is REF GT is variant HOMGT i i t HET OVERALLGT is variant HET GT is variant HET OVERALL

  • De novo mutations of SETBP1 cause Schinzel-Giedion syndrome

    Exome sequencing used to elucidate the causative mutations for a dominant Mendelian disorder Exomes of 4 affected individuals were enriched using gSureSelect All Exon kit and subjected to SOLiD sequencing Achieved 65-72% on target and ~ 85% of targeted bases at 10x depth (average depth at 43x) A number of prioritization steps resulted in 12 unknown A number of prioritization steps resulted in 12 unknown variants in 2 genes

    One gene, CTBP2, was determined to be a false positive p The second candidate, SETBP1, contained 4 different variants in a 11 bp region (affects 3 out of 4 highly consecutive amino acids)

    Sequencing in another 11 clinical samples confirms Sequencing in another 11 clinical samples confirms SETBP1 mutations in all samples

    Nature Genetics 2010

  • SureSelect All Exon Publications

    d t and many more to come

  • SureSelect Human All Exon 50Mbmost comprehensive coverage in the market

    SureSelect All Exon 50Mb Brand N Brand I

    CCDS (11/2010) 99 5% 98 4% 95%CCDS (11/2010) 99.5% 98.4% 95%

    RefSeq (11/2010) 99.0% 98.2% 91%

    GENCODE v4 97.0% ? ?

    target regions size 50Mb 44 Mb 62 Mb (?)

    bait (probe) length 120 80-85 95

    Insert size 150-200 bp 150-250 bp 300-400 bp

    Hyb. Time 24 hrs 72 hrs 24 hrs + 24 hrs

    Seq. output required 4-5 Gb ~ 5 Gb 10-12 Gb

  • Advantages of SureSelect All Exon Sequencing

    Average human exon size is ~ 150 bp

    SureSelect protocol recommends DNA shearing size to be 150-200 bp, and sequencing reads at 2x76 bp, matching nicely with average exon size

    ~ 150 bp

    with average exon size

    An exome DNA library with 300-400 bp shearing size results in off-target sequencing and additional sequencing cost

    Exon

    Exon2x76 bp

    and additional sequencing cost

    SureSelect All Exon Kits requires only ~ 5Gb sequencing, allowing more exomes per run and reducing sequencing cost accordingly asExon

    Exon

    2x76 bp

    2x150 bp

    and reducing sequencing cost accordingly as sequencing is the most expensive part of the workflow

  • Expanding Exome Design into Animal ModelsExon definition derived from Ensembl + RefSeqExon definition derived from Ensembl RefSeqDesigned against mm9 reference from UCSCNumber of genes covered: 24,306Number of exons: 221,784T t l i f d i 50 MB

    Mouse All Exon

    90%100%

    Performance Mouse All Exon Design with 5Gb sequencing

    Total size of design: 50 MB

    50%60%70%80%

    %

    0%10%20%30%40%

    0%% Reads On-

    Target +/- 200bpUniformity (3/4

    mean with upper tail)

    % Bases 1X Coverage

    % Bases 10X Coverage

    % Bases 20X Coverage

    Promega C3H DBA PWK 15NIH36a 15NIH49a

  • Multiplexing Option for SureSelect All Exon Kit

    HiSeq2000 GAIIx SOLiD 5500xl SOLiD 4

    Read length Output Output Output (nano)Output (micro) Outputg p p (nano) (micro) p

    PE (2x76 bp) < 150 Gb < 45 GbPE (75/35 or

    50/25 b ) < 270 Gb

  • Cost-effective Options using SureSelectXT System

    Introduction to SureSelectXT Target Enrichment System

    SureSelectXT All Exon Kits and MultiplexingSureSelect All Exon Kits and Multiplexing

    SureSelectXT Targeted Kits and Multiplexing

    Custom capture kitsp

    Human Kinome kits (DNA capture & RNA capture)

    Bravo Automation for Library Preparation and Capture

  • eArray web/eArray XD for SureSelect Custom Design

    eArray/eArray XD are free tools to design and ordercustom Microarrays and SureSelect libraries

    customer is the owner of a custom design SureSelect enrichment on eArray web features:

    search existing designs/baits download design filesdownload design files share designs upload custom bait designs add supplemental baits to catalogue content

    eArray XD is a desktop version with extra flexibility support bait design on custom genome support multiple genome builds of same speciesspecies has companion SureSelect Quality Analyzer

  • SureSelect Custom Design in eArray WebsiteSearch by gene accession number, chromosomal location

  • SureSelect Custom Design in eArray Website

    1. Enter a name for the design job.

    2. Design Strategy: To use the parameters previously optimized for general bait tiling, leave the checkmark on

    this option. To change the parameters, uncheck this option.