An efficient approach to BAC based assembly of complex genomes

Abstract

Background: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success.

Results: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes.

Conclusions: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

Keywords: 7DS; Assembly; BAC; Next-generation sequencing; SASSY; Saccharum spp; Triticum aestivum.

Fig. 1

Optimal coverage for assembly. Assembly sizes vs coverage for each of the 11 sugarcane BACs. Assembly sizes peak at 450x and level off despite increase in coverage beyond 1500x

Fig. 2

Mummer plot of assemblies of single BACs A, B, C, E against pooled BACs of ABCE

Fig. 3

BES mappings on contigs of simulated pool (ABCE). Clones A, C and E have forward (M13_For) and reverse (SP6_Rev) BES (A01_M13_For, A01_SP6_Rev, C01_M13_For, C01_SP6_Rev, E01_M13_For, E01_SP6_Rev) respectively correctly mapped. Clone B had no BES available but 120 bp sequences from cloning vector ends (FOR and REV) were used to identify contig ends of clone B

Fig. 4

Distribution of no of contigs and scaffolds per BAC for 96 BAC pools

Fig. 5

Distribution and orientation of MP insert sizes on E coli (a), contigs (b) and scaffolds (c) of 96 wheat BAC pools. Y axis (MP read counts in log scale), X axis (insert sizes). Correctly orientated MP reads with orientation RF (< –, – >) are shown in green, shadow library MP reads mapping with orientation FR (– >, < –) are shown in orange and chimeric MP reads mapping with orientation FF (– >, – >) and RR (< –, < –) are shown in blue