Computational protocol: Development of microsatellite markers and detection of genetic variation betweenGoniozuswasp populations

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Protocol publication

[…] Microsatellite-enriched genomic libraries were created essentially according to , with the final sequencing step performed using barcoded adaptors and a 1/16th run of non-titanium reagents Roche 454 pyrosequencing (as part of a mixture of 9 different libraries) ( www.454.com ). The generated Fasta files were separated in silico to identify the individual libraries, and those for G. legneri and G. nephantidis were searched for microsatellite motifs using the MISA.pl script ( http://pgrk.ipk-gartersleben.de/misa/misa.html ). Primer pairs flanking the simple sequence repeats were designed either by Primer 3 ( ) and/or WebSat ( ) for G. legneri ( ) and for G. nephantidis ( ). Primers were synthesized by Eurofins MWG Operon ( www.operon.com ) with a forward primer 5’ extension consisting of the M13 sequence to allow fluorescent labeling of the final product through a three primer reaction ( ), and prepared to 1000×concentration using Sigma-Aldrich ( www.sigmaaldrich ) molecular biology grade water (to create primer stocks of 200 pmol/µL). After vortexing and spinning, tubes were placed on ice for 30 min. Primers were kept in a freezer at -20°C, and to produce a 10×primer stock, 5µL of the 1000×stock was mixed with 495 µL sterile distilled water for both forward and reverse primers in separate tubes on ice. The third primer (M13;TGTAAAACGACGGCCAGT-3’) was ordered from Sigma-Aldrich and labeled with dye D4 (blue; WellRed dyes). […]

Pipeline specifications

Software tools MISA, WebSat
Application WGS analysis
Organisms Caenorhabditis elegans