Library preparation
The library preparation protocol consisted of (i) digestion using the
aforementioned restriction enzymes (ii) ligation with one of 48 unique
inline barcoded adapters compatible with the restriction site overhang,
(iii) manual sample pooling, (iv) DNA purification (QIAquick PCR
Purification Kit followed by SPRIselect paramagnetic beads), (v) 62 bp
narrow size-selection targeting fragments of 280– 342 bp in length
(BluePippin, Sage Science) and (vi) a PCR (polymerase chain reaction)
amplification step where one of two multiplexing index primers was
added. Indexed libraries were pooled together and loaded onto flow cells
for 150- bp paired-end sequencing on an Illumina NovaSeq 6000 platform.