DNA purification and sequencing

For Sanger sequencing, plasmid DNA recovered from embryos was transformed into XL1-Blue competent Escherichia coli and purified by alkaline lysis. Sanger sequencing was performed by Eton Bioscience Inc., using a primer approximately 200 bp from the I-SceI recognition site.

For high-throughput amplicon sequencing, approximately 300 bp of sequence flanking the I-SceI site was amplified from recovered plasmids by PCR with Q5 polymerase (NEB) for 19 cycles using an Eppendorf Vapo Protect thermocycler with a pooled set of primers containing one, two or three random bases at the 5′ end. AMPure bead purification was performed on the PCR products and the purified DNA was subjected to a second PCR to attach indices for amplicon sequencing. A final AMPure purification step was performed to remove all products less than 100 bp. The samples were pooled with 5% PhiX DNA and sequenced on an Illumina MiSeq platform using a version 3 chip with 2 × 300 paired-end reads.