Targeted Seq of dog blood
Library was prepared with Ion AmpliSeq Library kit2.0 (4475345) with custom designed panel of AmpliSeq primers panel design IAD165539, 404 amplicons in 2 pools, 61.19kb panel size, 96% coverage. To run multiple samples on the same chip we used IonExpress barcode adapters (kit#4471250 and #4474009). DNA was amplified on GeneAmp PCR system 9700 Dual 96-well thermal cycler from Applied Biosystems, using flowing protocol: Initial Hold  99*C-2min followed by  21cycles  @99*C-15sec, 60*C-4min and final  Hold 10*C inf. PCR product was purified with Agencourt AMPure XP kit (A63881 Beckman Coulter, Indianapolis, Indiana). Library was quantified using real time PCR  with  Ion Library TAQMAN Quantitation kit 44688022 on (Applied Biosystems ViiA7 Real Time PCR System) instrument to allow for optimal final dilution of library for template preparation on OneTouch OT2 instrument with Ion 540 Kit OT2 (A27753). The ISPs enrichment and purification was performed on Ion One Touch2 ES. Purified ISPs were analyzed on Ion Torrent personal Genome Machine using S5 instrument with 540 Kit OT2 (A27753) and 540 Chip Kit (A27766).  Data was collected and analyzed using Torrent Server (4462616) with Torrent 5.6.0. Final analysis of sequence data was performed using combination of software:  Variant Caller v.5.6.8-1, IGV5.01 (0). The Canfam3.fasta reference sequence was used for analysis.
411652-WB