RACA is similar, though it also requires paired-end sequencing data to aid scaffolding. For example, Chromosomer and MUMmer’s “show-tiling” utility leverage pairwise alignments to a reference genome for contig scaffolding and have been used to scaffold eukaryotic genomes. Finally, the analysis requires deep sequencing coverage and therefore can be expensive and compute-intensive.Īside from reference-free approaches, there are also a few tools available for reference-guided scaffolding. Also, because this process relies on the alignment of short Hi-C sequencing reads to the draft assembly, small and repetitive contigs with little or conflicting Hi-C link information often fail to be accurately scaffolded. Principally, Hi-C data are noisy, and Hi-C-based scaffolders are prone to producing structurally inaccurate scaffolds. Though Hi-C has been widely adopted, there remain challenges that can impede the ability to form accurate chromosome-scale pseudomolecules with Hi-C alone. Also, because misassemblies may be observed by visualizing Hi-C alignments, Hi-C can be used for validation and manual correction of misassemblies. According to the relative density of such Hi-C links between pairs of contigs, contigs can be ordered and oriented into larger scaffolds, potentially forming chromosome-length pseudomolecules. Paired-end Hi-C sequencing reads are aligned to the assembly, and mates which align to different contigs (Hi-C links) are recorded. In particular, Hi-C has recently been shown to be a practical and effective resource for chromosome-scale scaffolding. This includes a large class of technologies such as mate-pair sequencing, Bacterial Artificial Chromosomes (BACs), Linked Reads and chromatin conformation capture such as Hi-C. Furthermore, acquiring a genomic map can be expensive, time-consuming, or otherwise intractable depending on the species and the type of map.Īnother reference-free method for pseudomolecule construction involves the use of long-range genomic information to scaffold assembled contigs. However, contigs not implicated in any alignments will fail to be scaffolded, which can result in incomplete scaffolding. This process involves aligning the genomic map to a sequence assembly and scaffolding contigs according to the chromosomal structure indicated in the map. One popular reference-free scaffolding approach is to anchor genome assembly contigs to some variety of genomic map, such as an optical, physical, or linkage map. Two common approaches have been used to achieve chromosome-scale assemblies, namely, reference-free (de novo) and reference-guided approaches. Thus, there is a need for simplified and faster approaches to scaffold fragmented genome assemblies into chromosome-scale pseudomolecules. However, lagging behind the current speed and cost of generating long-read sequencing data are genome assemblers, which are still unable to resolve complex repeats and related structural variants that are widespread in eukaryotic genomes. Such analyses can include structural variations that are notoriously difficult to detect using short-read sequencing. Current long-read sequencers are now able to produce over one terabase of long reads per week, presenting the opportunity for detailed pan-genome analysis of unprecedented scale. Assemblies using these technologies in a variety of plant and animal species have consistently reported contig N50s over 1 Mbp, while also reconstructing higher percentages of target genomes, including repetitive sequences. Long-read single-molecule sequencing technologies commercialized by Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) have facilitated a resurgence of high-quality de novo eukaryotic genome assemblies.
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