Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies

Abstract

Background: New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from 'finished'. Updating multiscaffold drafts to chromosome-level status can be achieved through experimental mapping or resequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. Results:We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

A chromosome-scale assembly of the major African malaria vector Anopheles funestus

“…From this, an isofemale line was grown and DNA extracted from the adult females for sequencing with PacBio and Hi-C. A total of 46 single-molecule real-time (SMRT) cells of PacBio RSII sequencing using the P6-C4 chemistry were run by the core facility at the Icahn School of Medicine at Mount Sinai (New York, NY), resulting in 173× coverage (assuming a 250-Mbp genome size). A previous study generated 70× coverage of the same colony using the older PacBio P5-C3 chemistry sequencing [46]. These older data were combined with the additional 173× coverage, totaling 60.95 Gb of long-read data in 10.93 million sequences (average length 5.6 kb, N50 read length 8.4 kb) and an estimated total coverage of 234×.…”

“…This identified a mis-join of chromosomes 3R and X, which was manually corrected. Additional manual curation using mapped transcripts, fluorescence in situ hybridization (FISH) probes [46], and comparison to AfunF1 scaffolds identified a few additional inversion errors in the scaffolds, mainly on distal 2L. Visual inspection of the Hi-C data showed clear signatures of scaffolding error.…”

“…We next evaluated the structural accuracy of the AfunF1 and AfunF3 assemblies by measuring their agreement with the raw PacBio reads. The intermediate assembly AfunF2 [46] was assembled before collection of all PacBio and Hi-C data and so was deemed redundant and excluded from these analyses. When compared to the raw data, the AfunF3 primary assembly had fewer called structural differences (insertions, deletions, duplications, and inversions) than AfunF1 (Table 2).…”