Discovery and genotyping of structural variation from long-read haploid genome sequence data

Abstract

In an effort to more fully understand the full spectrum of human genetic variation, we generated deep single-molecule, real-time (SMRT) sequencing data from two haploid human genomes. By using an assembly-based approach (SMRT-SV), we systematically assessed each genome independently for structural variants (SVs) and indels resolving the sequence structure of 461,553 genetic variants from 2 bp to 28 kbp in length. We find that >89% of these variants have been missed as part of analysis of the 1000 Genomes Project even after adjusting for more common variants (MAF > 1%). We estimate that this theoretical human diploid differs by as much as ∼16 Mbp with respect to the human reference, with long-read sequencing data providing a fivefold increase in sensitivity for genetic variants ranging in size from 7 bp to 1 kbp compared with short-read sequence data. Although a large fraction of genetic variants were not detected by short-read approaches, once the alternate allele is sequence-resolved, we show that 61% of SVs can be genotyped in short-read sequence data sets with high accuracy. Uncoupling discovery from genotyping thus allows for the majority of this missed common variation to be genotyped in the human population. Interestingly, when we repeat SV detection on a pseudodiploid genome constructed in silico by merging the two haploids, we find that ∼59% of the heterozygous SVs are no longer detected by SMRT-SV. These results indicate that haploid resolution of long-read sequencing data will significantly increase sensitivity of SV detection.

Copy Number Variation Disorders

“…Whole genome sequencing (WGS) and analysis pipelines developed for variation detection have become increasingly robust over the past several years enabling the detection of thousands of novel CNVs and other structural variations [10–12]. Furthermore, the recent application of novel, long-read sequencing (77) and genome mapping (78) technologies have greatly enhanced the discovery of structural and copy number variations in whole-genome assays. Thus, the ongoing development and application of novel, high-throughput sequencing and mapping technologies holds great promise for the comprehensive detection of all forms of variation, especially CNVs, which will likely increase the diagnostic yield in patient cohorts.…”

Long-read genome sequencing identifies causal structural variation in a Mendelian disease

“…Another challenge comes in the form of structural variation, and although SRS has been very successful in the discovery of single nucleotide and small insertion-deletion variation, recent findings suggest we have greatly underestimated the extent and complexity of structural variation in the genome. 3,4 …”

New advances in sequence assembly

“…This includes detailed analysis of highly repetitive satellite sequence in flies (Khosta et al 2017) and birds (Weissensteiner et al 2017), paving the way for functional studies in areas of the genome not previously accessible. Long-read assembly is even revealing new variation in the human genome, and Huddleston et al (2017) highlights the importance of long-read sequencing and haplotype resolution for accurate structural variant detection.…”