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Comprehensive structural and copy-number variant detection with long readsAaron M. Wenger, Armin Töpfer, Yuan Li, Luke HickeyPacific Biosciences, 1305 O'Brien Drive, Menlo Park, CA 94025

Variant Detection with HiFi Reads

Existing long read variant calling methods rely on de novo assembly or spanning reads to detect variants. These methods are effective for SVs but miss many CNVs that involve long segmental duplications.

Copy-number variant (CNV)calling with pbsv CNVs in HG001 and COLO829T

PacBio highly accurate, long reads (HiFi reads) comprehensively detect variants in the human genome, including in difficult repetitive regions.

References1. Wenger AM, Peluso P et al. (2019). Accurate circular consensus long-read sequencing

improves variant detection and assembly of a human genome. Nat Biotechnol. doi:10.1038/s41587-019-0217-9.

2. Zook JM et al. (2019). An open resource for accurately benchmarking small variant and reference calls. Nat Biotechnol. 37(5):561-566.

3. Zook JM et al. (2019). A robust benchmark for germline structural variant detection. bioRxiv. doi:10.1101/664623. [Preprint]

4. Chaisson MJP et al. (2019). Multi-platform discovery of haplotype-resolved structuralvariation in human genomes. Nat Commun. 10(1):1784.

5. https://github.com/PacificBiosciences/pbsvThank you to David Scherer, Kristin Robertshaw, and Pamela Bentley Mills for poster production support.

Ref GCAGGCAGCGACTACGTACGCTAACAGCGATCTCAGAlt GCAGGCAGCGACTACGTCCTCTAACAGCGATCTCAG

SNVs

Indels

SVs

99.97% Precision99.95% Recall

99.10% Precision98.86% Recall

Precision and recall, as measured against the Genome in a Bottle (GIAB) benchmarks2,3, is high for single nucleotide variants (SNVs), indels, and structural variants (SVs).

Figure 1. Accurate PacBio HiFi reads detect variants in difficult-to-map exons of the disease gene STRC1.

Ref GCAGGCAGCGACTACGTACGCTAACAGCGATCTCAGAlt GCAGGCAGCGACTACGT-CGCTAACAGCGATCTCAG

Figure 2. PacBio SNV calling performance for HG002 with 32-fold HiFi coverage (Rowell, poster 1866/W).

Figure 3. PacBio indel calling performance for HG002 with 32-fold HiFi coverage.

"We determined that 57% and 15% of the copy number variable bases within segmental duplications detected by dCGH and Genome STRiP, respectively, were not in contigs resolved by de novo assembly [of long reads]."– Chaisson et al. 2019 (ref. 4)

We extended the PacBio SV caller, pbsv5, to detect CNVs using a combination of read clipping and depth.

Determine genome-wide coveragemedian coverage of non-gap

positions at mapping quality 60

Identify candidate CNV breakpointspositions with multiple clipped reads

Evaluate coverage between adjacent candidate breakpoints

calculate z-score vs Poisson expectation

pbsv CNV algorithm

Figure 5. PacBio read coverage is Poisson distributed in autosomes for (a) samples from GIAB and (b) the normal sample from a tumor/normal pair (provided by WP Kloosterman). A tumor sample shows CNV regions of reduced and increased coverage. (c) To call CNVs, pbsvidentifies candidate CNV breakpoints with multiple clipped reads and then evaluates read depth between adjacent breakpoints compared to the genome-wide typical coverage.

a b

c

candidate breakpoint

candidate breakpoint

HG001PacBio

reads

Segdups

Repeats

GRCh37 chr6:103,727,761-103,773,116 (45 kb)

25 kb

96.26% Precision94.93% Recall

RefAlt

Figure 4. PacBio SV calling performance for HG002 with 32-fold HiFi coverage.

HG001PacBio

reads

Segdups

Repeats

GRCh37 chr19:50,579,869-50,659,719 (80 kb)

HG001pbsv CNV

HG001PacBio

reads

Segdups

Repeats

HG001pbsv CNV

GRCh37 chr6:220,626-410,470 (189 kb)

Genes

Genes

Copy number Copy number

Tota

l CN

V le

ngth

(Mb)

CoverageCoverage

100

bp w

indo

ws

100

bp w

indo

ws

HG001 COLO829T (tumor)

segdup segdup

unique unique

Figure 6. pbsv CNV calls in HG001 in (a) segmentally duplicated (segdup) sequence and (b) unique sequence.

a

b

Figure 7. Genomic distribution of pbsv CNV calls. (a, b) Most CNVs in the "healthy" genome HG001 involve segdups. (c, d) The tumor genome COLO829T has many more CNVs than HG001 and most fall outside of segdups.

Summary

• PacBio HiFi reads comprehensively detect variants in a human genome.

• The pbsv variant caller identifies CNVs in HiFi reads using read clipping and depth signatures.

b

a

d

cGIABbenchmark

DeepVariantPacBio callset

3,047,8371,5711,030

GIABbenchmark

DeepVariantPacBio callset

459,4815,2834,329

GIABbenchmark

DeepVariantPacBio callset

9,196489357

540 6 12 18 24 30 36 42 48 60 900 10 20 30 40 50 60 70 80 1000%

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90 1 2 3 4 5 6 7 8 ≥10 90 1 2 3 4 5 6 7 8 ≥100

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HG001HG002HG005

COLO829NCOLO829T