Sequencing and Library Preparation Protocol

Sequencing was performed at the University of Arizona Genetics Core following previously described methods [31 (link)]. Briefly, 2 μg of DNA was sheared to 500 bp by ultrasonication on a Covaris S2. Samples were end-repaired, A tailed and ligated to adaptors Ind_Ad_T and Ind_Ad_B by NEBnext DNA library prep kit. PCR enrichment of TN5 junctions was performed with primers Tn-FO and Adapt-RO using Kapa HotStart HiFi (Roche) using thermocycling conditions as follows; 95° C for 5 minutes, 94° C for 45 seconds, 56° C for 1 minute, 72° C for 1 minute for 22 cycles, and a final extension at 72° C for 10 minutes. Samples were purified using a MagBio HighPrep PCR cleanup kit (MagBio Genomics) and amplicons from 100 to 600 bp were captured using a 2% agarose cassette on a Blue Pippin instrument (Sage Science). Amplicons were indexed using a Nextera V2 dual index kit (Illumina) and purified by MagBio HighPrep PCR cleanup. Purified, indexed amplicons were then quantified on a Qbit fluorometer, and sequenced on an Illumina MiSeq instrument as 75 bp paired end reads. Sequencing was repeated on these libraries in 3 separate runs to generate enough reads for analysis, and the reads were merged by sample after initial demultiplexing.