Limitations of next-generation genome sequence assembly

Abstract

High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics analyses and studies of genome evolution.

Genomic insights into salt adaptation in a desert poplar

“…Because of the limitations of next-generation sequencing for complex genome assembly 13 and the high levels of polymorphism found in this non-domesticated and open-pollinated species ( Supplementary Fig. S1), we employed a newly developed fosmid-pooling strategy 14 to sequence and assemble the P. euphratica genome (Table 1 and Supplementary Methods).…”

Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis

“…Despite our development and use of this high-resolution zebrafish aCGH platform, CNVs smaller than ∼4 kb and other types of genetic variants (i.e., balanced rearrangements and mobile elements) remain undiscovered in zebrafish. Further analyses to uncover these remaining structural genomic variants in the genome may include the use of next-generation sequencing, which would provide nucleotide-level breakpoint information and delineate which mechanisms predominate in CNV formation in the different strains (35,36). Using complementary technologies to meticulously identify and accurately genotype all forms of genetic variants will ultimately limit the variability in phenotypic outcomes resulting from epistatic effects of background genetic variants.…”

Reconstructing complex regions of genomes using long-read sequencing technology

“…While these methods systematically improve chromosomal contiguity across the genome (as measured by N50 contig length), they fail to accurately assemble the most complex regions of segmental duplications (Burton et al 2013). Regions targeted by our approach are frequently missing or grossly misassembled by whole-genome shotgun sequence assembly using either capillary or next-generation sequencing platforms (Alkan et al 2011b), still requiring highquality sequencing of large-insert clones to correctly resolve. Analysis of the mouse and human genomes suggests that these typically correspond to 300-500 regions (;140-150 Mbp) per genome, including in some cases almost entire chromosomes, such as the Y chromosome (Hughes et al 2012).…”

“…Complete high-quality sequence assembly remains a difficult problem for the de novo assembly of genomes (Alkan et al 2011b;Church et al 2011;Salzberg et al 2012). Finishing of the human and mouse genomes involved selecting large-insert BAC clones and subjecting them to capillary-based shotgun sequence and assembly (English et al 2012).…”