2bRAD seq of Astatotilapia burtoni: Adult female Fin clip
DNA was extracted from fin clips and fry using Maxwell 16 Tissue DNA Purification kit (Promega, USA) and then purified using Zymo DNA Clean & Concentrator kit (Zymo Research, USA) according to the manufacturers instructions. We then prepared sequencing libraries according to Wang (2012) (we used version 2bRAD_protocol_may15_2017_nnrw, the most up-to-date detailed protocol is available at https://github.com/z0on/2bRAD_denovo). Briefly, a type IIB restriction enzyme BcgI (New England Biolabs) was used to digest DNA into uniform 36 base pair fragments. Adaptors with unique molecular identifiers (UMI) ligated to the fragments barcoded only on the 3 end before being stored overnight at 4C. The ligase was then heat-inactivated with a 10-minute incubation at 65C. Samples were then pooled with 12 different 3 barcodes and amplified before a final purification step of the pooled libraries for the band at 160-180 base pairs using the Pippin Prep (Sage Science, USA) protocol. Libraries were sequenced on the Illumina HiSeq 2500 platform (Illumina, USA) at UT Austins Genomic Sequencing and Analysis Facility generating 418 million reads (2.9 million reads per sample on average).