PacBio HiFi Reads Improve Genome in a Bottle Variant Calling and Phasing
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- PacBio HiFi reads are long (>10 kb) and accurate (>99%). HiFi reads are available now for HG002 and soon for HG001 and HG005. - HiFi reads will be useful for comprehensive variant detection and phasing. Plans are outlined to apply HiFi reads to structural variant benchmarking and expand small variant calling to difficult regions.
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PacBio HiFi Reads Improve Genome in a Bottle Variant Calling and Phasing
- 1. For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019 by Pacific Biosciences of California, Inc. All rights reserved. PacBio Circular Consensus Sequencing of Genome in a Bottle samples Aaron Wenger 2019-03-28
- 2. PACBIO DATASETS FOR GIAB SAMPLES Wang, Olson, et al. (2019). bioRxiv. doi:10.1101/562611 Sample Sequencer CLR Datasets HG001 / NA12878 PacBio RS II 44-fold Ashkenazim Trio PacBio RS II 70-fold for HG002, 30-fold for HG003 and HG004 Chinese Trio Sequel System 60-fold for HG005, 30-fold for HG006 and HG007 200 6040 Read length (kb) Coverage(cumulative) 0 10 20 30 40 50 HG005 HG006 HG007
- 3. PACBIO CONTINUOUS LONG READS (CLR) NGS PacBio CLR Raw Reads IGV “Quick Consensus”
- 4. PACBIO CIRCULAR CONSENSUS SEQUENCING (CCS) Double-stranded DNA Ligate adapters Anneal primer and bind DNA polymerase Sequence Generate consensus HiFi read Subreads (passes) Subread errors Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025
- 5. PACBIO CIRCULAR CONSENSUS SEQUENCING (CCS) Double-stranded DNA Ligate adapters Anneal primer and bind DNA polymerase Sequence Generate consensus HiFi read Subreads (passes) Subread errors Passes 5 10 15 200 30 0 10 20 40 50 Accuracy(Phred) Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025 Read accuracy improves with more passes
- 6. HIFI READS ARE LONG AND ACCURATE Wenger, Peluso, et al. (2018). bioRxiv. doi:10.1101/519025 10 15 20 50 60 Read accuracy (Phred) Read length (kb) 403020 5 0 100,000 200,000 300,000 0 100,000 200,000 ReadsReads Avg. 13.5 kb Avg. 99.8% (Q27)
- 7. HIFI READS ARE LONG AND HIGHLY ACCURATE
- 8. HG002 HIFI DATASETS Coverage Average Read Length 30-fold 9.6 kb 28-fold 13.5 kb - Reads on SRA and NIST FTP - Alignments on NIST FTP Richard Hall, Paul Peluso, Yufeng Qian, David Rank, Billy Rowell https://bit.ly/2THv47q (NIST FTP)
- 9. Sequel System (1M) Sequel II System (8M) Subread Yield 318 Gb CCS Yield 16 Gb CCS Accuracy 99.8% Read length (kb) 0 50 100 150 200 250 300 Yieldperunitreadlength(kb) 0 25 50 100 150 175 75 125 SEQUEL II SYSTEM
- 10. Sequel System (1M) Sequel II System (8M) Subread Yield 318 Gb CCS Yield 16 Gb CCS Accuracy 99.8% Read length (kb) 0 50 100 150 200 250 300 Yieldperunitreadlength(kb) 0 25 50 100 150 175 75 125 Accuracy(Phred) 5 10 15 200 30 0 10 20 40 50 Sequel (1M) Passes 30 0 10 20 40 50 85 15 200 10 Passes Sequel II (8M) SEQUEL II SYSTEM
- 11. PLANS FOR NEXT QUARTER Sample Sequencer CCS Dataset HG002 Sequel System 30-fold with 9.6 kb reads HG002 Sequel System 28-fold with 13.5 kb reads HG002 Sequel II System 30-fold with 11.0 kb reads HG001 Sequel II System 30-fold with 10 kb reads HG005 Sequel II System 30-fold with 10 kb reads Submitted In Progress Primo Baybayan, Shreya Chakraborty, Alicia Yang
- 12. APPLICATIONS OF HIFI READS FOR GENOME IN A BOTTLE Structural variant benchmark -HG002: HiFi structural variant callset rivals multi-technology benchmark -Extend to HG001, HG005 Small variant benchmark (v4α for all samples) -Expand to difficult regions -Correct mistakes Phasing variants
- 13. VARIANT CALLING AND PHASING FOR STRC
- 14. SUMMARY bioRxiv 519025 doi:10.1101/519025 Baylor – Medhat Mahmoud, Fritz Sedlazeck Dana-Farber – Heng Li Chinese Academy of Agricultural Sciences – Jue Ruan DNAnexus – Chen-Shan Chin, Arkarachai Fungtammasan Google – Andrew Carroll, Pi-Chuan Chang, Mark DePristo, Alexey Kolesnikov Johns Hopkins – Michael Alonge, Michael Schatz Max Planck Dresden – Gene Myers NIH/NHGRI – Sergey Koren, Adam Phillippy NIST – Nathan Olson, Justin Zook PacBio – Greg Concepcion, Richard Hall, Paul Peluso, Yufeng Qian, David Rank, William Rowell, Armin Töpfer, Aaron Wenger Saarland University – Jana Ebler, Tobias Marschall -PacBio HiFi reads are long (>10 kb) and accurate (>99%). -HiFi reads are available now for HG002 and soon for HG001 and HG005. -HiFi reads will be useful for comprehensive variant detection and phasing.