Translocation panel, donor ND597, ABE-edited, 20x
Custom DNA target enrichment panel (Twist Biosciences; 474 probes, ~320 bp regions centered on the ABE on-target base, covering 56 candidate ABE target regions genome-wide, 22 relevant to this study, 99.55% design coverage). Library prep at GENEWIZ/Azenta: enzymatic fragmentation, end-repair, 3'-adenylation, adapter ligation, limited-cycle PCR (pre-capture), hybridization with biotinylated baits, streptavidin bead capture, post-capture amplification/indexing. Sequenced on Illumina MiSeq, 2x250 bp paired-end, ~10,000x target depth. Reads aligned to hg38 with BWA, processed per GATK Best Practices (Picard). On-target ABE editing efficiency assessed with LoFreq; Cas9 INDEL formation with CRISPResso2. Translocations detected via custom Python (pysam v0.23.3) + R pipeline: chimeric reads identified from supplementary alignment (SA) tags within a 500 bp window of each target site center; translocation frequency per site pair = unique chimeric reads / geometric mean of the two sites' coverage (%), cross-sample-normalized to reads per million on-target coverage; analyzed separately for 10- and 20-target designs; Mock (Cas9 or ABE8.3, no sgRNA) samples establish baseline spurious chimeric-read levels.