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Targeted Sequencing Panels on GIAB
Benchmark GenomesMegan Cleveland, Ph.D.
Applied Genetics Group - NIST
Genome in a Bottle Consortium WorkshopSeptember 15, 2016
Purpose - Goals• To perform targeted sequencing of GiaB reference materials (and
candidates)• Using commercial assays
• Further characterization of GiaB with assays of interest (clinical relevance) • Compare to high confidence regions• Identify/mitigate any discordant base calls• Potentially add new high confidence sequence information to the materials
Working in coordination with Justin and Marc
Samples for Targeted Sequencing
NA12878
Ashkenazi Trio RM 8392
Asian Trio
GM24143GM24149
GM24385NIST RM 8391
GM24695GM24694
GM24631NIST RM 8393
Caucasian FemaleNIST RM 8398
7 Individuals3 replicates
Selection Targeted Sequencing Panels
Ion AmpliSeq™ Pharmacogenomics PanelIon AmpliSeq™ Cancer Hotspot Panel v2Ion AmpliSeq™ Comprehensive Cancer PanelIon AmpliSeq™ Inherited Disease PanelIon AmpliSeq™ Exome Panel HID-Ion AmpliSeq™ Identity PanelHID-Ion AmpliSeq™ Ancestry Panel
TruSight OneTruSight HLATruSight RNA Pan-CancerTruSight MyeloidTruSight CancerTruSight Tumor 15TruSight CardioTruSight Inherited Disease
A starting point – input is welcome!
Overlap between TruSight and AmpliSeq IDPs
TruSight Inherited Disease Panel550 genes
AmpliSeq Inherited Disease Panel328 genes
454 96 232
Regions of interest are captured by hybridization to
biotinylated probes and isolated by magnetic pulldown.
Regions of interest are amplified using >10,000
primer pairs
Comparing the Inherited Disease Panels to the GIAB truth set
Data Analysis
SequenceReplicate 1 IDP VCF
Generate VCF with corresponding commercial Software
GIABVCF
IDP VCF
Compare the VCF files(ideal would be 100% overlap)
SequenceReplicate 2 IDP VCF
Generate VCF with corresponding commercial Software
GIABVCF
IDP VCF
Compare the VCF files(ideal would be 100% overlap)
SequenceReplicate 3 IDP VCF
Generate VCF with corresponding commercial Software
GIABVCF
IDP VCF
Compare the VCF files(ideal would be 100% overlap)
VCF from AmpliSeq or TruSight IDP
GIAB VCF
Intersection of GIAB and IDP bed files
GIABTruth
bed file
Inherited DiseasePanelbed file
New BedFile
Intersection of the Two Bed Files
~90% of each IDP covered in GIAB bed file
Examine Discordant Results in IGV• Vcfeval + Hap.py reports false negatives and false positives, with
respect to the GIAB VCF• The alignments in those locations are examined in IGV to determine
why the result is discordant
Source: broadinstitute.org
Acknowledgements• Applied Genetics Group
• Pete Vallone• Kevin Kiesler• Lisa Borsuk
• Genome Scale Measurements Group• Justin Zook• Marc Salit• Jennifer McDaniel• David Catoe• Lindsay Vang
Please email me with any questions or comments
