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

Genome Res. 2016 Nov 28. pii: gr.214007.116. [Epub ahead of print]


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. 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 when compared to 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 datasets 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 approximately 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.


Huddleston J1, Chaisson MJ2, Meltz Steinberg K3, Warren W3, Hoekzema K2, Gordon DS2, Graves-Lindsay TA3, Munson KM2, Kronenberg ZN2, Vives L2, Peluso P4, Boitano M4, Chin CS4, Korlach J4, Wilson RK3, Eichler EE5.