science wwebinar seriesebinar series dna target … slides...sdfsdfsdf make genomic dna fragment...
TRANSCRIPT
![Page 1: Science WWebinar Seriesebinar Series DNA Target … slides...sdfsdfsdf Make Genomic DNA Fragment Libraries Agilent Microarray ‐synthesis 120‐mer oligonucleotides ‐convert to](https://reader035.vdocuments.us/reader035/viewer/2022071107/5fe167886f4cfa544815d5b7/html5/thumbnails/1.jpg)
Sponsored by:
Participating Experts:
Daniel Turner, Ph.D.Wellcome Trust Sanger Institute,Cambridge, UK
Webinar SeriesWebinar SeriesScienceScienceDNA Target DNA Target 10 June, 200910 June, 2009
Brought to you by the Science/AAAS Business Office
Kelly Frazer, Ph.D.Scripps Genomic MedicineSan Diego, CA
Enrichment StrategiesEnrichment Strategies
www.opengenomics.com/SureSelect
![Page 2: Science WWebinar Seriesebinar Series DNA Target … slides...sdfsdfsdf Make Genomic DNA Fragment Libraries Agilent Microarray ‐synthesis 120‐mer oligonucleotides ‐convert to](https://reader035.vdocuments.us/reader035/viewer/2022071107/5fe167886f4cfa544815d5b7/html5/thumbnails/2.jpg)
Daniel J TurnerHead of Sequencing Technology Development
Wellcome Trust Sanger Institute
DNA Target EnrichmentStrategies – bringing efficiencies
to genome sequencing
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Target enrichment strategies
PCR
on array
in solution
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Target enrichment strategies
PCR
on array
in solution
• Design primers that are specific for the region of interest
• Amplify
• Sequence
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XR
1,438 samples
57 populations
Population sequencing of ACTN3
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The α-actinin-3 deficiency trade-off:
Compared to R577 homozygotes, R557X homozygotes have:
• lower muscle strength and mass
• reduced capacity for rapid energy generation
MacArthur et al. 2007. Nature Genetics 39:1261-1265MacArthur et al. 2008 Hum Mol Genet 17:1076-86
• increased endurance capacity
• increased fatigue recovery
• enhanced muscle metabolic efficiency
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Acoustic shearing
96-well library prep
ACTN3 CTSF
25 kb
Quail et al. (2008) Nat. Methods 5, 1005-1010
SPRI bead clean-ups
Custom adapters and barcoded PCR primers
Sequencing Strategy
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lanes 1,3,5,7 lanes 2,6,8
Sequencing Strategy
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Uniformity of coverage
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Fragment 10
• Uniformity is governed by the accuracy of pooling
• 80% with a coverage within 2-fold range of the median
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• 99.9% accuracy for genotype calling
• 63 high-confidence SNPs identified, 27 of them novel and 23 rare.
• Analysis of non-European HapMap samples and HGDP samples ongoing.
Results
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Target enrichment strategies
PCR
on array
in solution
• Limit of 5–20 kb per PCR
• Difficult to multiplex, optimise and normalise
• Uses a lot of DNA
• Expensive if multiplexing
• But very effective
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Target enrichment strategies
PCR
on array
in solution
• Hybridise sample DNA to target-specific probes on a microarray
• Wash to remove background
• Elute
• Sequence
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Target enrichment strategies
PCR
on array
in solution
• Hybridise sample DNA to target-specific probes in solution
• Capture probe / target
• Wash to remove background
• Elute
• Sequence
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gDNA Fragmentation
Target size: 100-300bpTarget size: 100-400bp
• Shorter fragments hybridize more efficiently
• Optimized settings give tighter distribution of fragment sizes
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Library purification
SPRI beads: easily automated
allow elution in a wider variety of buffers
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PCR and GC bias
Without PCR prior to hybridization
a. b.10 30 40 50 60
GC content (%)
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Percentile of unique sequence ordered by GC content
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Percentile of unique sequence ordered by GC content
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GC content (%)
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With PCR prior to hybridization
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• Completeness: % of target bases covered by >= 1 sequence read
• Specificity: % of sequences mapping to target regions
• Uniformity: variation in coverage
Evaluation parameters
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Completeness
On array ~ 98.6% of targeted bases
In solution ~ 99.5% of targeted bases
PCR =< 100% of targeted bases
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Specificity
On array up to 70% on target
In solution up to 80% on target
PCR up to 100% on target
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On array 90% of CTR at 30x
In solution 95% of CTR at 30x
90%
95%
100%
0 10 20 30Coverage (-fold)
% o
f CTR
bas
es 14M7.5M6.8M6.5M6.2M5.8MArray 6.5M
Sequence uniformity
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%GC vs %Coverage
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Target enrichment strategies
PCR
on array
in solution
• enables large-scale projects, which would not be realistic with PCR
• Not easily scalable
• Requires expensive hardware
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Target enrichment strategies
PCR
on array
in solution
• enables large-scale projects, which would not be realistic with PCR
• Simple & relatively rapid to perform
• Scalable & easily automated
• Uses least DNA
• Requires expensivehardware
• No whole exome set available commercially
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AcknowledgementsLira MamanovaCarol Scott
Iwanka KozarewaDaniel MacArthurChris Tyler-SmithQasim AyubLiz Huckle
Alison CoffeyEleanor HowardAarno Palotie
Wellcome Trust Sanger Institute
Emily LeProustFred Ernani
Agilent Technologies
Tom AlbertHeike FieglerGreg McGuiness
Nimblegen
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Sponsored by:
Participating Experts:
Daniel Turner, Ph.D.Wellcome Trust Sanger Institute,Cambridge, UK
Webinar SeriesWebinar SeriesScienceScienceDNA Target DNA Target 10 June, 200910 June, 2009
Brought to you by the Science/AAAS Business Office
Kelly Frazer, Ph.D.Scripps Genomic MedicineSan Diego, CA
Enrichment StrategiesEnrichment Strategies
www.opengenomics.com/SureSelect
![Page 26: Science WWebinar Seriesebinar Series DNA Target … slides...sdfsdfsdf Make Genomic DNA Fragment Libraries Agilent Microarray ‐synthesis 120‐mer oligonucleotides ‐convert to](https://reader035.vdocuments.us/reader035/viewer/2022071107/5fe167886f4cfa544815d5b7/html5/thumbnails/26.jpg)
Enrichment of sequencing targets from the human genome
Kelly A Frazer, PhDDirector, Genomic BiologyScripps Genomic Medicine
June 10, 2009
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genomic DNA
select regions
What is targeted sequencing?
Define sequence targets
Target enriched samples
Sequence
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Next‐Gen Sequencing
• Low costs for generating raw, per nucleotide sequence, ($0.00001 per base).
• Best suited for generating large amounts of raw sequence data per sample, (109nucleotides per day).
Still too costly and too low through‐put to perform whole‐genome sequencing for on many different DNA samples
Why perform targeted sequencing?
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To efficiently use current technologies for population‐based sequencing studies, it is necessary to enrich for specific loci in the human genome.
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Population Sequence Studies
• Sequence‐based association studies
Healthy elderly cohort versus individuals with age‐related diseases
• Functional annotation of genomic intervals
9p21 interval associated with CAD and T2D
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• PCR – enriches target sequences with high specificity but difficult to scale
• Hybridization based methods – long oligonucleotides in solution allow for efficient capture of ~3.5 Mb of sequence targets
• Microdroplet PCR – encapsulation of PCR reactions allows for simultaneous amplification of ~4,000 targeted elements
Sample enrichment methods
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Important parameters • Efficiency of assay design
– The fraction of targeted base pairs for which an assay can be designed
• Specificity of target enrichment– The fraction of high quality reads that map directly on the targeted sequences
• Coverage uniformity across targeted sequences– If coverage differs greatly then one has to sequence deeply to adequately cover underrepresented bases
• Reproducibility across technical replicates & samples
• Systematic allelic biases resulting in drop‐out effects– Errors of this nature result in high rates of incorrectly called heterozygous variant sites
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Target Enrichment by Solution Hybridization
sdfsdfsdf Make Genomic DNA Fragment Libraries
Agilent Microarray
‐ synthesis 120‐mer oligonucleotides
‐ convert to biotinylated RNA capture probes
‐ hybridization with DNA
‐ capture and wash
‐ elution and PCR amplify
‐ sequence targeted sequences
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3.6 Mb of Targeted Sequences
• 624 genes– 9,215 exons
– 4,886 evolutionarily conserved sequences (ECS)
– total 3.2 Mb of sequence
• 3 Contiguous Regions– 9p21: 196 kb
– APOE: 100 kb
– 8q24.21: 125 kb
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Probe design efficiency
(a)
(b)
genes
Repeat Mask
Probes
Chr9
CDKN2ACDKN2BAS
CDKN2BC9orf53
21950000 21960000 21970000 21980000 21990000 22000000 22010000 22020000 22030000 22040000 22050000 22060000 22070000 22080000 22090000 22100000 22110000 22120000 22130000
CDKN2A
CDKN2BASCDKN2B
21960000 21965000 21970000 21975000 21980000 21985000 21990000 21995000 22000000
FOXO1 gene
Repeat Mask
ECS Block
Probes
Chr13
ECS Signal
• 622 genes – CDS (97%) UTR (88%) ECS (86%)
• Three genomic intervals – 37% to 55%
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Specificity of target enrichment
38.6% map directly on target47.8% map on or near target (+/‐ 150 bp)
Percent of base pairs corresponding to filtered reads
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Coverage uniformity across targeted sequences
Normalized coverage – divided the observed coverage of each base by the mean coverage of all targeted bases
88.4% of all bases fell within ¼ to 4 times the mean coverage
98.3% of all bases covered by at least one read
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Reproducibility of coverage
Technical replicates r2 ~0.95
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Variant calling accuracycomparison to microarray genotypes
~ 4,100 SNPs
QS >= 30 detection rate = 93% concordance rate = 99.3%
No systematic allelic biases
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Solution hybridization‐based method is well suited for the enrichment of loci in the mega‐base‐pair scale from the human genome for population sequence studies
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Microdroplet PCR Workflow
Primer library – up to 4000 different elements
Fragmented genomic DNA template
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Primer design efficiency
• 47 genes – 435 exons– 29 from ENCODE intervals
– 8 TRP channel superfamily
– 11 deep venous thrombosis
• 457 amplicons of varying sizes (119‐956 bp) and GC content (33‐74%)
Successfully design PCR assays for all exons
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Specificity of target enrichment
• 78% of filtered reads successfully mapped to a targeted amplicon
• Off target reads aligned across genome in a random fashion ‐ suggesting that background sequence is due to non‐specific genomic DNA carryover rather then from off‐target amplification
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Coverage uniformity across targeted sequences
Normalized coverage – divided the observed coverage of each base by the mean coverage of all targeted bases
89.6% of all bases fell within ¼ to 4 times the mean coverage
99.6% of all bases covered by at least one read
Only one ampliconcompletely failed
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Reproducibility of coverage
Sample to sample r2 ~0.96
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Variant calling accuracycomparison to microarray genotypes
~ 450 SNPs
QS >= 30 detection rate = 97.6% concordance rate = 99.1%
Accuracy was similar in ENCODE versus non‐ENCODE interval variants and between samples of African and European ancestry indicating that allelic biases are mimimal
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The microdroplet PCR process is extremely efficient with almost 100% of all primer pairs successful. The data generated is well suited for performing population‐based sequence studies.
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Selecting a method
• Study design– Known functional elements or entire intervals
– Total amount of targeted sequences
– Number of samples
• Sequencing Technology
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AcknowledgementsSTSI/Scripps Genomic Medicine
Ryan Tewhey
Kazu Nakano
Wendy Wang
Sarah Murray
Olivier Harismendy
Eric Topol
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Sponsored by:
Participating Experts:
Daniel Turner, Ph.D.Wellcome Trust Sanger Institute,Cambridge, UK
Webinar SeriesWebinar SeriesScienceScienceDNA Target DNA Target 10 June, 200910 June, 2009
Brought to you by the Science/AAAS Business Office
Kelly Frazer, Ph.D.Scripps Genomic MedicineSan Diego, CA
Enrichment StrategiesEnrichment Strategies
www.opengenomics.com/SureSelect
![Page 51: Science WWebinar Seriesebinar Series DNA Target … slides...sdfsdfsdf Make Genomic DNA Fragment Libraries Agilent Microarray ‐synthesis 120‐mer oligonucleotides ‐convert to](https://reader035.vdocuments.us/reader035/viewer/2022071107/5fe167886f4cfa544815d5b7/html5/thumbnails/51.jpg)
Look out for more webinars in the series at:
www.sciencemag.org/webinar
For related information on this webinar topic, go to:
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