June 1, 2021

Comprehensive variant detection in a human genome with PacBio high-fidelity reads

Author(s): Rowell, W. and Peluso, P. and Harting, J. and Qian, Y. and Wenger, A. and Hall, R. and Rank, D.

Human genomic variations range in size from single nucleotide substitutions to large chromosomal rearrangements. Sequencing technologies tend to be optimized for detecting particular variant types and sizes. Short reads excel at detecting SNVs and small indels, while long or linked reads are typically used to detect larger structural variants or phase distant loci. Long reads are more easily mapped to repetitive regions, but tend to have lower per-base accuracy, making it difficult to call short variants. The PacBio Sequel System produces two main data types: long continuous reads (up to 100 kbp), generated by single passes over a long template, and Circular Consensus Sequence (CCS) reads, generated by calculating the consensus of many sequencing passes over a single shorter template (500 bp to 20 kbp). The long-range information in continuous reads is useful for genome assembly and structural variant detection. The higher base accuracy of CCS effectively detects and phases short variants in single molecules. Recent improvements in library preparation protocols and sequencing chemistry have increased the length, accuracy, and throughput of CCS reads. For the human sample HG002, we collected 28-fold coverage 15 kbp high-fidelity CCS reads with an average read quality above Q20 (99% accuracy). The length and accuracy of these reads allow us to detect SNVs, indels, and structural variants not only in the Genome in a Bottle (GIAB) high confidence regions, but also in segmental duplications, HLA loci, and clinically relevant “difficult-to-map” genes. As with continuous long reads, we call structural variants at 90.0% recall compared to the GIAB structural variant benchmark “truth” set, with the added advantages of base pair resolution for variant calls and improved recall at compound heterozygous loci. With minimap2 alignments, GATK4 HaplotypeCaller variant calls, and simple variant filtration, we have achieved a SNP F-Score of 99.51% and an INDEL F-Score of 80.10% against the GIAB short variant benchmark “truth” set, in addition to calling variants outside of the high confidence region established by GIAB using previous technologies. With the long-range information available in 15 kbp reads, we applied the read-backed phasing tool WhatsHap to generate phase blocks with a mean length of 65 kbp across the entire genome. Using an alignment-based approach, we typed all major MHC class I and class II genes to at least 3-field precision. This new data type has the potential to expand the GIAB high confidence regions and “truth” benchmark sets to many previously difficult-to-map genes and allow a single sequencing protocol to address both short variants and large structural variants.

Organization: PacBio
Year: 2018

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