Robust Benchmark Structural Variant Calls of an Asian Using State-of-the-Art Long-Read Sequencing Technologies

Abstract

The importance of structural variants (SVs) for human phenotypes and diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of these SV detection approaches, we established an Asian reference material by characterizing the genome of an Epstein-Barr virus (EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers, including 109× Pacific Biosciences (PacBio) continuous long reads (CLRs), 22× PacBio circular consensus sequencing (CCS) reads, 104× Oxford Nanopore Technologies (ONT) long reads, and 114× Bionano optical mapping platform, and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing, demonstrating the robustness of our SV calls. Combining trio-binning-based haplotype assemblies, we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions (CHCRs), which covered 1.46 gigabase pairs (Gb) and 6882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical research.

SVhawkeye: an ultra-fast software for user-friendly visualization of targeted structural fragments from BAM files

“…With SVhawkeye’s assistance, users can rapidly identify target regions across multiple disease samples, trio or pedigree samples, and more. For instance, SVhawkeye accurately detects balanced translocations, as demonstrated in various studies, including those referenced in PMC8804325 ( Pei et al, 2022 ), benchmark structural variant research ( Du et al, 2022 ), and population short tandem repeat counts, as verified in PMC9117641 ( Liu et al, 2022 ). SVhawkeye is well-suited for detecting structural variants in clinical samples generated from PacBio or Oxford Nanopore sequencing.…”

Haplotype-resolved assemblies and variant benchmark of a Chinese Quartet

“…Meanwhile, many variants in complex regions such as xMHC and segmental duplications were reported, which are difficult to detect using read-alignment based variant calling strategies. Another contribution of our benchmark is that, compared to previous studies [ 10 , 21 , 22 , 70 ], we extend the set of variant types to include complex structural variants and de novo mutations. Nevertheless, our benchmark also has several limitations.…”

“…Authoritative and comprehensive variant benchmarks are therefore crucial for precisely understanding genetic variation in clinical samples. During the past decades, many consortiums such as Genome in a Bottle [ 5 – 10 ] (GIAB), Sequencing Quality Control [ 11 – 19 ], and Illumina Platinum Genomes [ 20 ] have established many variant benchmarks and genomic reference materials [ 19 , 21 , 22 ]. These resources help the community evaluate their variant detection strategies.…”

Profiling the Genome-Wide Landscape of Short Tandem Repeats by Long-Read Sequencing

“…Sample LNT00178 was also sequenced with the Pacibio Sequel II platform. High molecular weight (HMW) DNA was extracted, and HiFi libraries were constructed using the SMRTbell Express Template Prep Kit v2 and SMRTbell Enzyme Clean Up Kit (PacBio) ( Du et al, 2021 ). Size selection was performed with SageELF and 15 kb fragments were chosen for sequencing with the Sequel II platform using 30 h movies.…”