, the protocol was specifically customized for cfDNA processing. Overall, based on the quality and the low quantity of starting material , library preparation was performed with specific adjustments, whereas the target enrichment was customized with the design of a small target panel.
Library preparation started directly with end-repair reaction by using NEBNext End Repair Module following the manufacturer’s protocol, therefore avoiding any fragmentation step. To minimize the loss of so short fragments )of low-input cfDNA, a clean-up step with an optimized ratio volume of AMPure XP beads was performed, without size selection.
The target of interest was defined starting from the identification of genes relevant for tumorigenesis, evolution and emergence of drug resistance in CRC. In detail, the genomic size of high-sensitivity capture LB panel is 59 kilobases for a total of 300 captured regions.
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