GBS Data Analysis for Tomato Genome Mapping

GBS data analysis was performed using the GBS discovery pipeline of TASSEL version 5.0 software [30 (link)]. The FASTQ and sample key files (containing the barcodes for each genotype) generated from raw sequence reads by the CASAVA 1.8.2 software package (Illumina Inc.) were used as input for processing in the pipeline. Before the analysis, 64-base reads were generated by trimming reads having the barcodes for each genotype followed by an ApeKI cut site using the FastqToTagCountPlugin of the pipeline. Reads with unidentified bases (N) were excluded from analysis. The barcoded sequence reads were collapsed into unique sequence tags with counts using the FastqToTagCountPlugin with default parameters with the exception that minimum number of times a tag must be present was set to 3. Tag count files that contained the sequence tags that passed the minimum count threshold of 3 were merged into a master file using the MergeMultipleTagCountPlugin. The master tags in FASTQ format generated by TagCountToFastqPlugin were aligned to the tomato S. lycopersicum reference genome using the bowtie2 plugin with default parameters [31 , 32 (link)]. SAMConverterPlugin generated the “Tags On Physical Map” (TOPM) file which contained information about the physical positions of the master tags which had the best unique alignments with the reference genome. In addition to the TOPM file, the “Tags by Taxa” (TBT) file that contained tag counts of each barcode generated by FastqToTBTPlugin was used for SNP calling according to the parameters of the TagsToSNPByAlignmentPlugin (Additional file 1: Table S1). SNPs were recorded in a HapMap file for each chromosome. MergeDuplicateSNPsPlugin was used to merge the duplicate SNPs. SNPs were filtered based on minimum Taxon Coverage (mnTCov: 0.01), minimum Site Coverage (mnSCov: 0.2), linkage disequilibrium with neighboring SNPs (hLD: TRUE), minimum R2 value for the LD filter [−mnR2]: 0.2, and minimum Bonferroni-corrected p-value for the LD filter [−mnBonP]: 0.005. A physical map of the identified SNPs was drawn using Mapchart software [33 (link)].