Illumina TruSeq Synthetic Long-Reads Empower De Novo Assembly and Resolve Complex, Highly-Repetitive Transposable Elements

Abstract

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5–18.5 Kbp with an extremely low error rate (0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.

Speciation with gene flow in equids despite extensive chromosomal plasticity

“…High-resolution structural characterization (34,35) will be required to fully explore the precise influence of chromosomal rearrangements on gene flow. For now, our study provides the first empirical evidence connecting gene flow and chromosomal rearrangements in equids.…”

Haplotype-phased synthetic long reads from short-read sequencing

“…In contrast to dilution-based synthetic read approaches [17][18][19], the intramolecular circularization step in our protocol makes it possible to prepare a library in a single tube. We further exploited this property to allow multiple samples to be combined and prepared in the same mixture, yielding considerable savings in cost and time.…”

“…We have shown that coverage is uneven across barcode groups, varying by up to two orders of magnitude (S9 Fig). As a result of their common reliance on long-range PCR, our method and the TruSeq Long Reads approach may exhibit bias against regions of the genome due to secondary structure or GC content, though we do not observe GC bias in the datasets presented here (S7 Fig). Synthetic read methods also fail to assemble regions containing short repeats, which can be accurately sequenced by true long read methods (McCoy et al 2014).…”

“…"True" long read technologies such as Pacific Biosciences SMRT sequencing and nanopore technologies [10,11] have proven invaluable in select applications, but require extensive computational error correction [12,13]. As an alternative, sample preparation protocols have emerged that enable long "synthetic" reads to be constructed from conventional short reads [14][15][16][17][18][19][20][21]. However, these approaches have been limited by generalizability, cost, throughput, or synthetic read length.…”

Read Clouds Uncover Variation in Complex Regions of the Human Genome

“…The Illumina TruSeq protocol is able to produce 10-kbp long reads, retaining the benefits of the highly accurate and cost-effective Illumina technology (Kuleshov et al 2014) and enabling human genome phasing (Kuleshov et al 2014) and de novo assembly of complex genomes (Voskoboynik et al 2013;McCoy et al 2014).…”