Direct determination of diploid genome sequences

By N. Weisenfeld, Vijay Kumar, Preyas Shah, D. Church, David B. Jaffe. bioRxiv, 2016

754 citations recorded by the source corpus.

Determining the genome sequence of an organism is challenging, yet fundamental to understanding its biology. Over the past decade, thousands of human genomes have been sequenced, contributing deeply to biomedical research. In the vast majority of cases, these have been analyzed by aligning sequence reads to a single reference genome, biasing the resulting analyses and, in general, failing to capture sequences novel to a given genome. Some de novo assemblies have been constructed, free of reference bias, but nearly all were constructed by merging homologous loci into single ‘consensus’ sequences, generally absent from nature. These assemblies do not correctly represent the diploid biology of an individual. In exactly two cases, true diploid de novo assemblies have been made, at great expense. One was generated using Sanger sequencing and one using thousands of clone pools. Here we demonstrate a straightforward and low-cost method for creating true diploid de novo assemblies. We make a single library from ~1 ng of high molecular weight DNA, using the 10x Genomics microfluidic platform to partition the genome. We applied this technique to seven human samples, generating low-cost HiSeq X data, then assembled these using a new ‘pushbutton’ algorithm, Supernova. Each computation took two days on a single server. Each yielded contigs longer than 100 kb, phase blocks longer than 2.5 Mb, and scaffolds longer than 15 Mb. Our method provides a scalable capability for determining the actual diploid genome sequence in a sample, opening the door to new approaches in genomic biology and medicine.

Subfields: Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/9bcfb860b26ebc4b1444bf00b013ed7f76afa573

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Citations in the corpus

Integrating Hi-C links with assembly graphs for chromosome-scale assembly (2018, background): “…clones (Venter et al., 1996; Gnerre et al., 2011), optical maps (Schwartz et al., 1993; Dong et al., 2013; Shelton et al., 2015), linked reads (Zheng et al., 2016; Weisenfeld et al., 2017; Yeo et al., 2017), or chromosomal conformation capture (Simonis et al., 2006) to order and orient unitigs.”
TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads (2020, neutral): “Inaddition,Super-nova[37](version2.”
WhatsHap: fast and accurate read-based phasing (2016, background): “It is sometimes also called haplotype assembly, but should not be confused with haplotype-resolved (de novo) genome assembly, which has recently also been explored [12, 13].”
Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies (2018, methodology): “…assemblers such as FALCON and Canu are available that will produce a haplotype-fused representation of a diploid genome [4, 6], and some assemblers such as FALCON Unzip and Supernova will go further to produce large phase blocks where both parent alleles are represented separately [4, 7].”
De novo assembly of haplotype-resolved genomes with trio binning (2018, neutral): “21).”
ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter (2016, positive): “The technology employs microfluidics to isolate large DNA fragments in partitions containing sequencing primers and a unique barcode, preparing a library that is compatible with Illumina paired-end sequencing (Weisenfeld et al. 2017).”