Simultaneous sequencing of genetic and epigenetic bases in DNA
For five-letter seq, cfDNA or sheared gDNA was mixed with 1 µl of spike-in control , 3.5 µl End Prep reaction buffer and 1.5 µl End Prep Enzyme Mix for end repair and A-tailing . The reaction was incubated at 20 °C for 30 min followed by 65 °C for 30 min. In the same mix, Adapter 1 was ligated using 3.75 µl of adapter 1, 0.5 µl of ligation enhancer and 15 µl of ligation master mix and incubated at 20 °C for 15 min.
To generate standard genomic libraries the KAPA Hyper Prep kit was used according to the manufacturer’s protocol with the following modifications. Using a Covaris M220 sonicator, 100 ng of genomic DNA was sonicated to ~250 bp in 50 µl sonication tubes and used in the experiment. For adapter ligation, standard TRUSeq adapters were used . Ampure beads were used instead of KAPA clean-up beads and the elution volume was reduced to 21 µl using library dilution buffer .
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