A COMPREHENSIVE VIEW OF THE GENOME - Unmappable regions - Segmental duplications and tandem repeats 5 Mb 3 Mb 10 Mb 1 bp SNVs ≥50 bp structural variants 1-49 bp indels Prior tech vs
A COMPREHENSIVE VIEW OF THE GENOME SMRT Sequencing provides even coverage across difficult to sequence regions of the genome. Almost no coverage with prior tech PacBio reads sequence straight through and detect variants, some falling in coding regions Prior tech PacBio reads STRC Haplotype 1 Haplotype 2
A COMPREHENSIVE VIEW OF THE GENOME - Long insertions - Events in repeat regions 5 Mb 3 Mb 10 Mb 1 bp SNVs ≥50 bp structural variants 1-49 bp indels Prior tech vs
HiFi reads pbmm2 Google DeepVariant variant calls (vcf) -DeepVariant learns error model of HiFi reads from training data -Improved precision and recall for both SNVs and Indels DeepVariant v0.9 was released today with training on Sequel II System Chemistry 2.0 and improved performance on all chemistries. 15-fold HiFi vs GIAB v3.3.2 benchmarks Sample SNV Recall SNV Precision Indel Recall Indel Precision HG001 99.1% 99.5% 94.1% 95.0% HG002 99.2% 99.5% 95.4% 96.6% HG005 99.4% 99.7% 97.0% 97.5% alignment variant detection WGS SMALL VARIANT CALLING WORKFLOW WITH DEEPVARIANT HAS HIGH PRECISION AND RECALL
15-FOLD HiFi READ COVERAGE RECOMMENDATION Wenger, A. et al., Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. 2019. Nature Biotechnology.
REDUCED ANALYSIS TIME WITH HIFI READS Compute times for de novo assembly of a human genome Data Type HiFi Reads Long Reads Input File Type CCS.FASTQ.GZ SUBREADS.BAM Input File Size (GB) 48 323 Read Correction Method CCS Analysis Pre-assembly Time to Results (Hours) Read Correction 17.5 43.5 Contig Assembly 13.7 18.9 Analyses run with PacBio recommended compute infrastructure ~31 hrs ~62 hrs Faster compute time, lower compute and storage costs
HIFI DATA NOW SUPPORTED BY FALCON-UNZIP https://github.com/PacificBiosciences/pbbioconda/wiki/Assembling-HiFi-data:-FALCON-Unzip3 -Native support for HiFi -Faster read tracking and polishing -Incorporating HiFi-specific overlapping options -Phasing algorithm improvements ✔ ✔ ✔
CONCLUSIONS -HiFi reads enable accurate, comprehensive detection of genetic variation. -We recommend using pbmm2 for alignment, DeepVariant for small variant detection, and pbsv for structural variant detection. -Improvements to the Sequel II chemistry and optimization of the FALCON workflow have improved the contiguity and accuracy of HiFi assemblies at a fraction of the compute time and compute costs of CLR assembly.
Small variant detection Andrew Carroll Pi-Chuan Chang Mark DePristo Richard Hall Alexey Kolesnikov Justin Zook, Justin Wagner, and the Genome in a Bottle Consortium Structural variant detection Armin Töpfer Aaron Wenger ACKNOWLEDGMENTS De novo assembly Sarah Kingnan Greg Concepcion Jim Drake Chris Dunn Richard Hall Jonas Korlach Zev Kronenberg Ivan Sovic Michelle Vierra Aaron Wenger Alicia Yang Greg Young
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