Read2Marker: a data processing tool for microsatellite marker development from a large data set.

Microsatellites, or simple sequence repeats (SSRs), are widely considered to be one of the best sources of DNA polymorphism that can be utilized for developing genetic markers (1). Recently, several methods for making microsatellite-enriched genomic libraries were reported (2,3) and, as a consequence, the random sequencing of a microsatellite-enriched genomic library becomes a possible strategy for the mass development of microsatellite markers. Despite the progress in microsatellite isolation, it remains timeconsuming and laborious to handle the large amounts of sequence data. A set of script programs, read2Marker, was developed as a solution to this problem (Figure 1). In cooperation with phred/ phrap (4), BLAST (5), Primer3 (6), and T-Coffee (7), read2Marker can process an unlimited number of raw output data files from an automatic sequencer. It can run on a MacOS X or Linux platform and generates a report on many aspects of the nonredundant microsatellite-containing clones such as whole sequence, microsatellite core sequences, and PCR primers. A set of trace data obtained by sequencing from both ends of a clone is base-called by phred and assembled by phrap. Based on quality values (QVs), the high QV part of the assembled sequence is used for further processing. When data in plain text format without QVs are to be processed, all of the nucleotides are automatically assigned a QV of 20 to disable the quality threshold aspect of the read2Marker program. Subsequently, a microsatellite core is screened in the clone sequence by a newly developed tool, srchssr2. Initially, srchssr2 searches for repeated sequences of four dinucleotide motifs (GA, GT, AT, GC) and 10 trinucleotide motifs (AAC, AAG, ACC, ACG, ACT, AGC, AGG, ATC, TAT, CGC) independently in the clone sequence, and the core with the highest repeat number is selected. Next, a core structure neighboring the selected longest core is rescreened. If the gap between these cores is shorter than a maximum gap length set by the user in advance, then the two cores and gap sequence are connected to make a new core sequence. These procedures are repeated, and finally the composite microsatellite core in the sequence is identified. Finally, a PCR primer set for amplifying the microsatellite core is designed using the Primer3 program. The algorithm for finding microsatellites with srchssr2 is quite simple, but it is efficient enough to find cores. It can find composite repeats composed of different motifs and extracts 5′and 3′-flanking sequences that will be used for a redundancy check in the next step. Several SSR-finding programs with more sophisticated algorithms such as Msatfinder (M.I. Thurston and D. Field, www.bioinf.ceh.ac.uk/msatfinder), TROLL (8), Sputnik (C. Abajian, espressosoftware.com/pages/sputnik. jsp), and Tandem repeats finder (9) are available, and some of them could be substituted for srchssr2 with some modification, especially in cases where screening for motifs of more than three nucleotides is required. In order to remove redundant sequences, a homology check is performed using the BLAST algorithm. Two outer sequences of the clone flanking the microsatellite core are independently set as query sequences and BLASTed to a database comprising unique clones. If at least one of the two core-flanking sequences does not provide a hit from the database, the clone is judged as unique and added to