Assembly and diploid architecture of an individual human genome via single-molecule technologies

Abstract

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

De novo assembly and phasing of a Korean human genome

“…We also extended into 72 of the 85 remaining gaps with the addition of 663 kb of sequence into 4.1 Mb. These locations, previously intractable using only short reads, commonly contained simple tandem repeats, as reported previously 6,9 . One example (Fig.…”

“…The final assembly after polishing with Illumina reads (Extended Data Fig. 4a) is characterized by marked contiguity that has not been achieved by non-reference assemblies of the human diploid genome [6][7][8] so far, and improves on the previous best 6 N50 length by 18 Mb ( Table 1). The largest 91 scaffolds, for example, cover 90% of the genome and 8 chromosomal arms are spanned by single scaffolds (Fig.…”

Improved maize reference genome with single-molecule technologies

“…Leaves were used to prepare high molecular mass DNA and optical genome maps were constructed as described above for B73. Structural variant calls were generated based on alignment to the reference map B73 v4 chromosomal assembly using the multiple local alignment algorithm (RefSplit) 32 . A structural variant was identified as an alignment outlier 32,49 , defined as two well aligned regions separated by a poorly aligned region with a large size difference between the reference genome and the map or by one or more unaligned sites, or alternatively as a gap between two local alignments.…”

“…Structural variant calls were generated based on alignment to the reference map B73 v4 chromosomal assembly using the multiple local alignment algorithm (RefSplit) 32 . A structural variant was identified as an alignment outlier 32,49 , defined as two well aligned regions separated by a poorly aligned region with a large size difference between the reference genome and the map or by one or more unaligned sites, or alternatively as a gap between two local alignments. A confidence score was generated by comparing the non normalized P values of the two well aligned regions and the non normalized log likelihood ratio 50 of the unaligned or poorly aligned region.…”

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

“…We used the diploid aware PacBio assembler FALCON 0.3.0 (ref. 32), which has recently been used to successfully assemble highly heterozygous genomes 33 , to assemble the F. cylindrus genome. The cut off for long reads was 2,000 bp.…”