Long-read sequencing and de novo assembly of a Chinese genome

Abstract

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

A reference genome of the Chinese hamster based on a hybrid assembly strategy

“…Recent studies have highlighted the improvements of SMRT-only assemblies compared to Illumina-only assemblies [Bickhart et al, 2017], [Gordon et al, 2016], [Jiao et al, 2017], [Zhang et al, 2016], [Shi et al, 2016]. Here we found that both the pooled Illumina assembly (with mixed read length) and the PacBio SMRT-only assembly resulted in substantially improved assembly statistics compared to the two published hamster genome assemblies (Table 1), with an order of magnitude fewer scaffolds and 2–4x larger N50 values.…”

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

“…New reference-quality sequence sources are needed, because generation of finished sequence from clone libraries is in significant decline due to cost and some remaining assembly gaps occur in regions recalcitrant to cloning. A growing collection of human genomes in INSDC databases, a prerequisite for any sequence that will contribute to the reference assembly, that were sequenced and assembled with new technologies are candidates for use in assembly improvement (Earl et al 2011;Vezzi et al 2012;Bradnam et al 2013;English et al 2015;Pendleton et al 2015;Seo et al 2016;Shi et al 2016;Zook et al 2016). However, WGS assembly sequences have historically not been considered reference quality, raising concerns about their use in reference genome assembly curation.…”

“…Alignments were performed and analyzed as described in the Supplementary Methods of Shi et al (2016). However, in contrast to the RefSeq transcripts, we evaluated coverage for the GENCODE data over the full transcript, rather than the CDS, because we did not have the CDS information.…”

“…The reference assembly is not just a substrate for alignment, but is also the coordinate system on which we annotate our biological knowledge. Several recently published individual human de novo assemblies have been favorably compared to GRCh38 with respect to continuity metrics, and although they each contain sequence not present in the reference assembly, none yet surpass the global quality of GRCh38 Steinberg et al 2014;Berlin et al 2015;Cao et al 2015;Pendleton et al 2015;Seo et al 2016;Shi et al 2016). Such assemblies are often suggested as sequence sources for use in closure of reference assembly gaps, whereas other studies have called for one or more individual genomes to replace the reference .…”

Investigation of repetitive sequences in the human genome

“…The scoring function of this approach is the likelihood of a pair of label intervals being similar [39]. These assembled contigs and alignments can be used in further hybrid assembly using sequencing reads from other technologies to yield an improved overall final assembly with longer N50( [40,41]). …”