title: MiSeq amplicon sequencing of tyr, insra, and insrb
design: Genomic DNA from individual larvae was extracted using 50mM NaOH and then neutralized with 1M Tris.HCl. The targeted regions of tyr, insra and insrb were amplified using gene-specific primer pairs in which the forward primer containing M13 promoter sequence. A subsequent round of PCR added a 6 bp barcode to the 5' end of the M13 sequence. All 49 samples received a unique barcode:    A1 AAAAAA  A2 AAAATT  A3 AAAAGG  A4 AAAACC  A5 AAATAT  A6 AAATTA  A7 AAATGC  B1 ATCCTA  B2 ATCCGC  B3 ATCCCG  B4 AGAAAG  B5 AGAATC  B6 AGAAGA  C1 TAACTC  C2 TAACGA  C3 TAACCT  C4 TATAAA  C5 TATATT  C6 TATAGG  D1 TTCTAG  D2 TTCTTC  D3 TTCTGA  D4 TTCTCT  D5 TTCGAT  D6 TTCGTA  E1 TGCGAG  E2 TGCGTC  E3 TGCGGA  E4 TGCGCT  E5 TGCCAC  E6 TGCCTG  F1 GACTAA  F2 GACTTT  F3 GACTGG  F4 GACTCC  F5 GACGAC  F6 GACGTG  G1 GCCTAC  G2 GCCTTG  G3 GCCTGT  G4 GCCTCA  G5 GCCGAA  G6 GCCGTT  H1 CAAAAC  H2 CAAATG  H3 CAAAGT  H4 CAAACA  H5 CAATAG  H6 CAATTC    After amplification, PCR products were purified using a QIAquick PCR purification kit (Qiagen) and products pooled together. The sequencing library was made from 100ng PCR products using the Ovation Ultraflow Library system (NuGen Inc.), quantified by qPCR (KAPA BioSystems).