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GIAB Sep2016 Lightning megan cleveland targeted seq
This document discusses targeted sequencing of Genome in a Bottle (GIAB) reference materials using commercial panels to further characterize the materials. Seven individuals from various GIAB materials will be sequenced in triplicate using various targeted panels, including cancer and inherited disease panels. The results will be compared to the high confidence GIAB regions to identify any discordant variants and potentially add new information. Overlap between different panels and to the GIAB regions will also be examined.
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GIAB Sep2016 Lightning megan cleveland targeted seq
- 1. Targeted Sequencing Panelson GIAB Benchmark Genomes Megan Cleveland, Ph.D. Applied Genetics Group - NIST Genome in a Bottle Consortium Workshop September 15, 2016
- 2. 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
- 3. Samples for TargetedSequencing NA12878 Ashkenazi Trio RM 8392 Asian Trio GM24143GM24149 GM24385 NIST RM 8391 GM24695GM24694 GM24631 NIST RM 8393 Caucasian Female NIST RM 8398 7 Individuals 3 replicates
- 4. Selection Targeted SequencingPanels Ion AmpliSeq™ Pharmacogenomics Panel Ion AmpliSeq™ Cancer Hotspot Panel v2 Ion AmpliSeq™ Comprehensive Cancer Panel Ion AmpliSeq™ Inherited Disease Panel Ion AmpliSeq™ Exome Panel HID-Ion AmpliSeq™ Identity Panel HID-Ion AmpliSeq™ Ancestry Panel TruSight One TruSight HLA TruSight RNA Pan-Cancer TruSight Myeloid TruSight Cancer TruSight Tumor 15 TruSight Cardio TruSight Inherited Disease A starting point – input is welcome!
- 5. Overlap between TruSightand AmpliSeq IDPs TruSight Inherited Disease Panel 550 genes AmpliSeq Inherited Disease Panel 328 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
- 6. Comparing the InheritedDisease Panels to the GIAB truth set
- 7. Data Analysis Sequence Replicate 1 IDPVCF Generate VCF with corresponding commercial Software GIAB VCF IDP VCF Compare the VCF files (ideal would be 100% overlap) Sequence Replicate 2 IDP VCF Generate VCF with corresponding commercial Software GIAB VCF IDP VCF Compare the VCF files (ideal would be 100% overlap) Sequence Replicate 3 IDP VCF Generate VCF with corresponding commercial Software GIAB VCF IDP VCF Compare the VCF files (ideal would be 100% overlap)
- 8. VCF from AmpliSeqor TruSight IDP GIAB VCF Intersection of GIAB and IDP bed files
- 9. GIAB Truth bed file Inherited Disease Panel bed file New Bed File Intersectionof the Two Bed Files ~90% of each IDP covered in GIAB bed file
- 10. Examine Discordant Resultsin 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
- 11. We welcome inputregarding: • Additional panels • Data analysis • Other Megan.Cleveland@nist.gov
- 12. Acknowledgements • Applied GeneticsGroup • 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 Megan.Cleveland@nist.gov