Manali Landfill Metagenome
1. Qualitative and Quantitative analysis of received metagenomic DNA: The quality and quantity of the received metagenomic DNA samples was checked on NanoDrop followed by agarose gel electrophoresis. 2. Preparation of 2 x 150 Meta Genome Library: The paired-end sequencing libraries were prepared from the QC passed metagenomic DNA samples using illumina TruSeq Nano DNA Library Prep Kit. Briefly, approximately250ng of DNA was fragmented by Covaris M220 to generate a mean fragment distribution of 350bp. Covaris shearing generates dsDNA fragments with 3' or 5'overhangs. The fragments were then subjected to end-repair followed by adapter ligation to the fragments. This strategy ensures a low rate of chimera (concatenatedtemplate) formation. The ligated products were size selected using AMPureXP beads. The size-selected products were PCR amplified with the index primer as described in the kit protocol indexing adapters were ligated to the ends of the DNA fragments, preparing them for hybridization onto a flow cell. 3. Quantity and quality check (QC) of library on Agilent 4200 Tape Station: The PCR enriched libraries were analyzed on 4200 Tape Station system (AgilentTechnologies) using high sensitivity D1000 Screen tape as per manufacturer instructions. 4. Cluster Generation and Sequencing: After obtaining the Qubit concentration for the libraries and the mean peak sizes from Agilent Tape Station profile, the PE illumina libraries were loaded onto NovaSeq6000 for cluster generation and sequencing. Paired-End sequencing allows the template fragments to be sequenced in both the forward and reverse directions on NovaSeq6000. The kit reagents were used in binding of samples to complementary adapter oligoes on paired-end flow cell. The adapters were designed to allow selective cleavage of the forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand was then used to sequence from the opposite end of the fragment.