Illumina Synthetic Long Read Sequencing Allows Recovery of Missing Sequences even in the “Finished” C. elegans Genome

Abstract

Most next-generation sequencing platforms permit acquisition of high-throughput DNA sequences, but the relatively short read length limits their use in genome assembly or finishing. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking. We evaluate the promise and deficiency of the long reads in these aspects using isogenic C. elegans genome with no gap. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences prevents pre-assembly of long reads in the relevant genomic region. Second, the reads are able to reliably detect missing but not extra sequences in the C. elegans genome. Third, the reads of smaller size are more capable of recovering repetitive sequences than those of bigger size. Fourth, at least 40 Kbp missing genomic sequences are recovered in the C. elegans genome using the long reads. Finally, an N50 contig size of at least 86 Kbp can be achieved with 24×reads but with substantial mis-assembly errors, highlighting a need for novel assembly algorithm for the long reads.

Specific down-regulation of spermatogenesis genes targeted by 22G RNAs in hybrid sterile males associated with an X-Chromosome introgression

“…We previously demonstrated that Illumina synthetic long reads not only are able to recover nonrepetitive sequences but also are capable of recovering most types of repetitive sequence except for those arranged in a long stretch of tandem repeats (Li et al 2015). To facilitate comparative analysis of expression of small RNAs, TEs, and protein-coding genes between wildtype and hybrid strains, we produced approximately 20× coverage of Illumina synthetic long reads for C. nigoni as described previously for a C. elegans genome assembly (Li et al 2015). Most of the reads are ∼10 kbp in length with a minimum size of 1.5 kbp.…”

“…In particular, the short reads are problematic for assembling contigs that are rich in repetitive sequences, which will prevent accurate annotation of these sequences, including TEs and some small RNAs that are to be addressed in this study. We previously demonstrated that Illumina synthetic long reads not only are able to recover nonrepetitive sequences but also are capable of recovering most types of repetitive sequence except for those arranged in a long stretch of tandem repeats (Li et al 2015). To facilitate comparative analysis of expression of small RNAs, TEs, and protein-coding genes between wildtype and hybrid strains, we produced approximately 20× coverage of Illumina synthetic long reads for C. nigoni as described previously for a C. elegans genome assembly (Li et al 2015).…”

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

“…This was due to more accurate assembly of large repetitive elements in the C. elegans genome, resulting in an improved genomic reference. It has recently been reported that the extent of some repetitive elements in the reference genome are truncated (Li et al 2015). The MinION-generated genome presented here expands repetitive elements throughout the genome, adding more than 2 Mb to the reference genome.…”

“…These assemblies are significantly more contiguous than those generated by Illumina Synthetic Long-Read Sequencing or by PacBio sequencing for the C. elegans genome. MinION-derived assemblies resulted in an assembly with an N50 contig size of 3.99 Mb compared to an N50 of 86 kb for Synthetic Long-Read Sequencing (Li et al 2015) and an N50 of 1.6 Mb for PacBio sequencing (https://github.com/PacificBiosciences/DevNet/wiki/ C.-elegans-data-set). Combining flow cell data improved the assemblies.…”

“…Even more problematic for genome assembly are local repeats that range from short repetitive sequences, such as homopolymeric G tracts (Zhao et al 2007), to large tandem repeats spanning tens of kilobases. Although much effort was invested in the reference genome, it was generated before the availability of longer single molecule sequence reads, and a recent study reports that some repeat regions may be truncated in the reference genome (Li et al 2015). Here, we report the MinION-derived sequencing and de novo assembly of two C. elegans genomes, a complete C. elegans reference genome and a strain with two complex chromosomal rearrangements.…”