Heliconius erato Sd NcoI-RAD mapping
Preparation of a RAD sequencing library preparation followed the canonical protocol (www.radseq.info) with the following adaptations. Genomic DNA from 25 [Sdnot/not] and 25 [Sdhim/him] F2 individuals from a single H. himera x H. erato notabilis family were dispatched equimolarly (50ng/individual) into two separate Sdnot/not and Sdhim/him DNA pools. Each pool was digested with the restriction enzyme NcoI, heat inactivated, and directly ligated to custom barcoded, Illumina Paired-End sequencing forward adapters ( Sdhim/him PE1 :5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[CAGTA]G ; 3’-TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[GTCAT]CGTACp ; Sdnot/not PE1 : 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT[CGTAG]G ; 3’-TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA[GCATC]CGTACp ; brackets denote five-nucleotide barcodes). The two multiplexed samples were then sheared to an average size of 400bp using a Covaris S220 shearer (Covaris Inc.), size selected to 300-600bp using an agarose gel extraction, and blunt ligated to a custom reverse adapter (PE2 : 5’- GTTCGTCTTCTGCCGTATGCTCTAGCCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAG*A ; 3’-ATCGTCACTCGGCATTCCTGCTGAACCGCTCTTCCGATCp ; asterisk denotes a phoshorothiate bond modification). The resulting library was then amplified by Touchdown PCR ( 5 cycles at 65C -1ºC/cycle ; 13 cycles at 60ºC ; each cycle including denaturation at 98ºC and extension at 72ºC) using the high-fidelity Phusion Taq polymerase (Finzymes) and custom amplification primers (PE1 : 5’- AATGATACGGCGACCACCG*A ; PE2 : 5’- CAAGCAGAAGACGGCATACGA*G ; asterisks denote phoshorothiate bond modifications), gel extracted between 300-700bp, and sequenced on a single lane of an Illumina Genome Analyser GAII generating 72bp Paired-End reads (Illumina Inc.) at the Institute for Integrative Genome Biology (University of California, Riverside, USA).
UNiversity of California, Irvine
Heliconius erato Sd NcoI-RAD PE-72bp