DIDA-Seq Custom Capture cfDNA Library Preparation v1

Dual Index Degenerate Adaptor Sequencing, or DIDA-Seq, an ultra-sensitive cell-free DNA sequencing approach for the detection and quantification of circulating tumor DNA in peripheral blood plasma at fractions below the background error-rate of traditional next-generation sequencing (NGS). DIDA-Seq combines unique molecular identifiers (UMIs) and patient-specific custom capture panels to routinely detect ctDNA with mutation allele frequencies at or below 0.02% (or 1 tumor derived DNA fragment in 5k normal fragments at a given genomic locus) with error-rates below 1 base in 10kb-50kb (Butler et al 2019, doi:10.1101/mcs.a003772; Boniface et al 2021, doi:10.3390/diagnostics11010073). This protocol describes the preparation of DIDA-Seq libraries using cell-free DNA, custom DIDA-seq library adaptors and blocking oligonucleotides, and custom hybridization capture panels. It uses reagents developed and manufactured by Roche, LTD and Integrated DNA Technologies, Inc with minimally modified versions of their methods.