Hyten et al. BMC Genomics 2010, 11:38 http://www.biomedcentral.com/1471-2164/11/38 rate in soybean for developing a successful GoldenGate assay from a validated SNP was 89%. Taking into account this 89% conversion rate, the production run Maq analysis method effectively had a SNP validation rate of 91.7%. This is very similar to the 92.5% valida- tion rate obtained from the initial Maq analysis method based upon two or more reads. This high validation rate matching the validation rate obtained with two or more reads was expected to some degree, given that only 63 of the 1,536 SNPs were predicted based upon a single short sequence read. Using the production run Maq assembly, we were able to target 1,536 SNPs (of the total 7,108 SNPs) onto the preliminary 6.5× scaffold assemblies to help improve the subsequent anchoring and orientation of the 8× scaffold assembly of soybean into the 20 pseudomolecule Glyma1.01 build [1]. We were able to target SNPs to scaffolds that heretofore could not be anchored using the Consensus Map 4.0. We were able to orient addi- tional scaffolds. Where possible, two markers were cho- sen at the ends of each unanchored or un-oriented scaffold. This led to the W82 × 468 genetic map having an uneven distribution of markers as they were clustered in regions where the Consensus Map 4.0 had few or no markers [Additional file 3]. Overall the initial use of the Consensus Map 4.0 followed by the subsequent use of the W82 × 468 high-resolution map, resulted in a whole genome sequence of soybean of which more than 97% is anchored to the genetic map [1]. The high resolution map ultimately provided markers for 335 of the 8× scaf- folds which totaled more than 872 Mb of the completed genome sequence. Many of these scaffolds contain mar- kers from the Consensus Map 4.0, but the high-resolu- tion map provided not only a rigorous confirmation of scaffold contiguity, but also ordered and oriented 23 of the 8× scaffolds for which no markers existed on the consensus map. Conclusions The availability of a diversity of both low- and high- multiplex SNP assay methods makes SNPs an ideal mar- ker for QTL mapping, association analysis, marker- assisted selection (MAS), and the construction of high density genetic maps for fine-mapping and cloning of agronomically important genes. We have demonstrated how next generation sequencing combined with high- throughput SNP detection assays can quickly discover a large number of SNPs that can then be used to create a high resolution genetic map. Further, such high-resolu- tion maps are critical for accurate placement of sequence scaffolds into chromosomal pseudomolecules. Page 5 of 8 Methods Reduced Representation Library Construction Seeds of the G. soja genotype PI 468916 were obtained from the USDA-ARS, Soybean Germplasm Collection courtesy of Dr. Randall L. Nelson. DNA was isolated from bulk leaf tissue of 10 to 15 plants as described by Keim et al. (1988). Four different blunt end restriction enzyme combinations each containing five different restriction enzymes each were tested as an effort to reduce the likelihood of repetitive sequence. A single combination of five enzymes resulting in the least band- ing in the 100 to 150 bp region of the restriction digest was selected. The restriction digestion of PI 468916 to create the RRL consisted of digesting a total of 50 μg of DNA with the combination of 30 units of HaeIII, 15 units of PsiI, 15 units of SspI, 30 units of RsaI, and 15 units of MslI. The restriction digestion was carried out overnight at 37°. The digested DNA was then run on a 2% agarose gel and the digestion products were excised from the gel in the 100 to 150 bp region. The QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) was used as per the manufacturer’s protocol to obtain a total of 4,300 ng of size selected DNA. The blunt-ended DNA fragments were provided to Illumina for sequencing on the Genome Analyzer (Illumina, Inc; San Diego, CA, USA). The sequence data obtained from Illumina con- tained 33 bp sequence tags in which every base is given a quality score that is similar to a Phred score and has a maximum value of 40 [12]. The Illumina sequence data have been deposited in the NCBI, Sequence Read Archive [GenBank:SRA010205]. SNP discovery Initially the GMAP method was used for SNP discovery from the sequence produced from the reduced represen- tation library with the Illumina Genome Analyzer. GMAP software [13] was used for alignment of the 33 bp PI 468916 Illumina Genome Analyzer reads with the Williams 82 preliminary 4× scaffold assembly for the discovery of putative SNPs. The following constraints were used in the filtering for the identification of SNPs: 1) short reads which contained a base with a quality score < 10 were eliminated, 2) reads were selected that had only one alignment to the Williams 82 reference sequence and had to have 32 or 33 matching nucleo- tides with no part of the sequence part of a repeat (as determined by GMAP, in the 3rd column of the BLAT- output PSL format), no “N” bases in the alignment, and no gaps present in the query sequence and no gaps in the reference sequence, 3) alignments that had any con- flicting nucleotides were eliminated, 4) SNPs where both flanking 25 nt regions were repetitive were eliminated (specifically, when both flanking regions matched other locations in the genome with two or fewer mismatches),