ARS | Publication request: Application of RAD LongRead sequencing for SNP discovery in sugarcane

Submitted to: Plant Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: January 17, 2011
Publication Date: January 17, 2011
Citation: Glynn, N.C., Nipper, R.W., Comstock, J.C. 2011. Application of RAD LongRead sequencing for SNP discovery in sugarcane. Plant Genome Conference Proceedings. 184.

Technical Abstract: The sugarcane (hybrid Saccharum spp.) genome presents a difficult challenge for SNP discovery and analysis due to its complex polyploid nature. This is compounded further due to the absence of a reference genome sequence. We report the discovery of SNPs in sugarcane through reductive sequencing and local sequence assembly using a combination of RAD (Restriction site Associated DNA) LongRead protocols and Illumina/Solexa paired-end sequencing chemistry. RAD libraries were processed by digesting genomic DNA from two genotypes, sugarcane cultivar CP 92-1167 and Saccharum spontaneum accession IND 81-146 with Sbfl followed by sequencing on an Illumina Genome Analyzer IIx. Raw sequence reads from each genotype were first coalesced into approximately 3.0 Mb of assembled sugarcane genomic sequence distributed across approximately 6,500 unique sequence contigs. The assemblies were aligned to the Sorghum bicolor reference genome and served as a framework for pairwise sequence alignment and marker discovery. A custom variant discovery pipeline based on sequencing depth observed at each SNP position enabled us to distinguish between putative simple SNPs and hemi-SNPs. Quantitative genotyping was performed for a selection of simple and hemi-SNPs using the Sequenom MassArray. Concordance was achieved between the putative SNP dosages observed in the alignments and the quantitative results observed in the genotyping assays. This result demonstrates the utility of the RAD genome complexity reduction technique and LongRead assembly approach as an efficient approach for SNP discovery in sugarcane.