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) ( ). 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 script ( ). 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 ( ) 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