Genotyping-By-Sequencing (GBS) Using a Semiconductor Sequencing Platform

The development of new sequencing technologies has greatly enhanced the discovery of SNPs. Using a reduced representation library strategy to sequence the restriction enzyme digested fragments, genotyping-by-sequencing (GBS) is becoming a popular genotyping tool in genetics studies and breeding applications. By multiplex using barcodes, the sequencing cost per sample is significantly low. The challenges, however, still exist when running GBS in Ion Proton, a semiconductor sequencing platform. The number of SNPs called from an Ion Proton run is very low. Moreover, after proportion present filtering, the number of SNPs called from Ion Proton is less than one tenth of that from Illumina HiSeq 2000.To investigate the relationship between sequencing depth and number of SNPs called, we constructed a library of 192 samples and ran the library multiple times to determine optimal sequencing depth for GBS SNP analysis. We compared two methods for merging duplicated sequencing data to increase the sequencing depth. We found that sequencing a library twice is a cost-effective choice for SNP analysis, which yields the acceptable number of SNP markers for mapping or genomic selection.