Dumas, M. D. | Search bioRxiv

bioRxiv2020

Genome assembly of a Mesoamerica-derived Phaseolus lunatus cultivar enables the identification and comparative genomic analysis of a complex resistance locus

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa and a resource for identification of potential resistance genes. Combined efforts of sequencing, linkage and comparative mapping analysis resulted in a 625 Mb genome assembly, as well as a better understanding of a locus underlying resistance to the current downy mildew race in the field.

genomics↗

bioRxiv2017

Clustering of Circular Consensus Sequences: Accurate Error Correction and Assembly of Single Molecule Real-Time Reads from Multiplexed Amplicon Libraries

BACKGROUNDTargeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical challenge for research and applications using HTS data, especially from long-read platforms, is errors arising from technological limits and bioinformatic algorithms.\n\nRESULTSA single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing.\n\nCONCLUSIONSC3S-LAA uses a novel processing algorithm for SMRT amplicon-sequence data that produces accurate consensus sequences and local sequence assemblies. The community standard long amplicon analysis module from Pacific Biosciences is prone to substantial errors that raise concerns about findings based on this pipeline. The method developed here removed this confounding bioinformatics source of error, allowing for the identification of limited instances of errors due to DNA amplification or sequencing.

bioinformatics↗