DNAseq of five generations of a selection experiment on two populations of Globodera pallida
DNAseq of five generations of a selection experiment on two populations of Globodera pallida
A maximum of 10,000 eggs was inoculated into two litre pots containing a 2.2 kg mixture of silver sand, kaolin, hydro grains and nutrients. To each of the pots, a potato piece containing a single shoot was added. To obtain sufficient starting material, prior to the selection experiment, AMPOP02 was propagated on the susceptible variety Desiree for two consecutive generations, resulting in AMpop02D2. AMPOP10 was propagated on Desiree for one generation, resulting in AMpop02D1.  For each generation, we aimed to save cysts to later use for DNA extraction.   J2 larvae collected after counting were frozen and stored at -80°C. These were lysed  and DNA was isolated using phenol/chloroform/isoamyl alcohol. DNA quantities were determined using Qubit Fluorometer (Invitrogen). Because we were dependent on how well the material was preserved after counting not all samples provided DNA of sufficient quantity and/or quality, 45 out of 60 samples were successfully sequenced. The DNA was sequenced at BGI Genomics (Hong Kong) using BGISEQ-500 with a median output of 295.4*106 reads of 100 bases (paired) per sample. Upon arrival the samples, integrity and purity were tested by gel-electrophoresis. Per sample 1 µg of DNA was fragmented by Covaris and fragments in the size ranges of 150-250 bp were selected which were subsequently quantified by Qubit. Fragments were end-repaired and 3' adenylated prior to adaptor-ligation to the 3' end. The fragments were amplified by PCR and purified with the Agencourt AMPure XP-Medium kit. DNA was quantified by Agilent 4200 TapeStation. Double stranded PCR products were circularized, and the circular DNA was used as input for sequencing in the BGISEQ-500 platform.