MANDI LANDFILL METAGENOME
1. Qualitative and Quantitative analysis of received metagenomic DNAThe quality and quantity of the received metagenomic DNA samples was checked onNanoDrop followed by agarose gel electrophoresis.2. Preparation of 2 x 150 Meta Genome LibraryThe paired-end sequencing libraries were prepared from the QC passed metagenomicDNA samples using illumina TruSeq Nano DNA Library Prep Kit. Briefly, approximately250ng of DNA was fragmented by Covaris M220 to generate a mean fragmentdistribution of 350bp. Covaris shearing generates dsDNA fragments with 3' or 5'overhangs. The fragments were then subjected to end-repair followed by adapterligation 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 inthe 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 StationThe PCR enriched libraries were analyzed on 4200 Tape Station system (AgilentTechnologies) using high sensitivity D1000 Screen tape as per manufacturerinstructions.1. Qualitative and Quantitative analysis of received metagenomic DNAThe quality and quantity of the received metagenomic DNA samples was checked onNanoDrop followed by agarose gel electrophoresis.2. Preparation of 2 x 150 Meta Genome LibraryThe paired-end sequencing libraries were prepared from the QC passed metagenomicDNA samples using illumina TruSeq Nano DNA Library Prep Kit. Briefly, approximately250ng of DNA was fragmented by Covaris M220 to generate a mean fragmentdistribution of 350bp. Covaris shearing generates dsDNA fragments with 3' or 5'overhangs. The fragments were then subjected to end-repair followed by adapterligation 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 inthe 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 StationThe PCR enriched libraries were analyzed on 4200 Tape Station system (AgilentTechnologies) using high sensitivity D1000 Screen tape as per manufacturerinstructions.4. Cluster Generation and SequencingAfter obtaining the Qubit concentration for the libraries and the mean peak sizes fromAgilent Tape Station profile, the PE illumina libraries were loaded onto NovaSeq6000for cluster generation and sequencing. Paired-End sequencing allows the templatefragments to be sequenced in both the forward and reverse directions onNovaSeq6000. The kit reagents were used in binding of samples to complementaryadapter oligoes on paired-end flow cell. The adapters were designed to allow selectivecleavage of the forward strands after re-synthesis of the reverse strand duringsequencing. The copied reverse strand was then used to sequence from the oppositeend of the fragment.