Novaseq pe150 instrument

The water samples (1000 mL) were sequentially filtered through 0.45 and 0.22 µm GE® polycarbonate filter membrane. The membranes from 0.22 µm were cut into small pieces in sterilized condition and DNA extraction was performed using an optimized CTAB method (Zhou et al. 1996 (link)). The DNA extractions were performed in triplicates and replicates were later pooled prior to metagenome sequencing. DNA quality and quantity was examined with a Thermo Scientific NanoDrop 3300 Fluorospectrometer (Thermofisher Scientific, USA). The extracted DNA was randomly sheared into short fragments and ligated with Illumina adapters to construct a library. The libraries were pooled, barcoded and subsequently shotgun sequenced on one lane of a flow cell using a 150 bp paired-end run on a NovaSeq PE150 instrument (Illumina) at Novo-gene (Hong Kong). The sequences were de-multiplexed using Cassava v.2.0 and FastQC was used for quality control checks on the sequence composition of paired-end raw reads. Trimmomatic v0.36 (Q-value ≤ 38; N >10 bp; reads overlap with adapter >15 bp) was employed to remove low quality bases and any adapter contamination.

Fresh leaves were cut into 2-cm pieces and fixed with 2% formaldehyde. Hi-C library construction was performed as previously described^{23 (link)}. In brief, genomic DNA was extracted, and the fixed chromatin was digested by DpnII, followed by a fill-in reaction with biotinylated nucleotides and proximity ligation. The DNA was purified and then sheared into ~ 350-bp fragments using a Covaris S220 device. The DNA fragments were subjected to blunt-end repair, A-tailing, Illumina paired-end adapter ligation and PCR amplification. The Hi-C library was sequenced on an Illumina NovaSeq PE150 instrument.