No. of occurrences of a particular 33-mer 500 plus 300-500 100-299 35-99 20-34 15-20 11-14 9-10 7-8 6 5 4 3 2 1 TOTAL 1,293 1,414 6,561 15,119 14,510 12,040 15,645 15,234 29,215 26,648 43,105 72,955 130,555 259,225 1,312,518 1,956,037 2,142,203 536,701 1,097,771 871,270 374,818 206,542 192,046 143,581 216,367 159,888 215,525 291,820 391,665 518,450 1,312,518 8,671,165 Hyten et al. BMC Genomics 2010, 11:38 http://www.biomedcentral.com/1471-2164/11/38 Page 3 of 8 Table 1 Numbers of occurrences of 33-mer Illumina Genome Analyzer sequence reads, numbers of 33-mers in each occurrence category as well as the total 33-mer reads and total bases in each occurrence category. No. of unique 33-mers No. of 33 base reads picked parameter sets with the general goals of predict- ing unique, polymorphic, well-supported SNPs, given constraints of available sequence and the need to gener- ate markers in certain marker-poor regions. The first SNP prediction method, for testing and vali- dation involved the use of GMAP software [13] for alignment of unique PI 468916 short sequence reads to the Williams 82, preliminary 4× scaffold assembly, with SNPs predicted from the alignments. The preliminary 4× scaffold assembly was a “test run” produced by the sequencing consortium for an initial assessment of assembly characteristics of the genome. At the same time, we used this early assembly to initially test and validate using next generation sequencing and different software for SNP-prediction. The GMAP alignment method, with stringent match criteria (using only high- quality reads, unique mappings, multiple-reads SNP sup- port; details in Materials and Methods), produced a total of 10,778 predicted SNPs. The validation set for the GMAP method consisted of 635 primer pairs designed to flank each candidate SNP. Of these 635 primer pairs, a total of 535 produced a sequence tagged site with high quality sequence surrounding the predicted SNP, and 456 of the 535 produced amplicons containing the pre- dicted SNP, which constituted an 85% validation rate. The second SNP prediction method employed was the Maq mapping and assembly software [14] to align unique PI 468916 short sequence reads to the prelimin- ary 4× scaffold assembly and predict SNPs from that assembly. The Maq analysis method, when used with selected parameters (minimum consensus-base quality of 20, unique read placement; details in Materials and Methods), produced a total of 25,047 predicted SNPs, each predicted using one or more short sequence reads aligned to one position within the genome. The first validation set for the Maq procedure consisted of 48 pri- mer pairs designed to flank SNPs predicted from two or more short sequence reads. Of these 48 primer pairs, a total of 40 produced a sequenced tagged site (STS) with high quality sequence surrounding the predicted SNP, and 37 of the 40 produced amplicons containing the predicted SNP, which translated into a 92.5% validation rate. We also tested the validation rate of predicting SNPs with a single short read. For this test, the Maq analysis method above was used, but without the requirement for multiple read support of predicted SNPs. A total of 48 primer pairs were designed to flank SNPs predicted from only one short sequence read. Of the 48 primer pairs tested, 43 produced an STS with high quality sequence surrounding the predicted SNP, and 34 of those 43 produced amplicons that contained the pre- dicted SNP, which resulted in a 79% validation rate. To identify SNPs best suited for GoldenGate assay design and for anchoring and orienting the preliminary 6.5× scaffold assembly, we used a modification of the Maq method described in the validation tests above. In this “production” run, we required a consensus score of at least 27, and that the flanking 120 bases be at least 2/ 3 non-repetitive sequence (see Materials and Methods for implementation). Because of the high (79%) valida- tion rate for SNPs called from single short sequence reads using Maq, these SNPs were also included in this dataset so that additional markers on more of the Total bases 70,692,699 17,711,133 36,226,443 28,751,910 12,368,994 6,815,886 6,337,518 4,738,173 7,140,111 5,276,304 7,112,325 9,630,060 12,924,945 17,108,850 43,313,094 286,148,445