Sample prep comparison for wastewater sequencing
This metagenomic library was generated from the filter fraction of a wastewater sample filtered through a 3-m membrane, replicate 2. Metagenomic Illumina libraries were prepared using the NEBNext Ultra DNA Library Preparation Kit (New England Biolabs, Cat. #E7370S) according to the manufacturer's protocol. Due to varying amounts of extracted DNA, the input ranged from 10 ng to less than 1 ng. DNA was fragmented using a Covaris E220, the ends made blunt, and adapters and indices added onto the ends of the fragments prior to PCR enrichment. For samples with inputs less than 10 ng of DNA, the number of PCR cycles was increased from the manufacturer's protocol of 10 to 15 cycles. AMPure XP beads (Beckman Coulter, Cat. #A63881) were used to clean up the libraries prior to elution in DNA Elution Buffer (Zymo Research, Cat. #D3004-4-10). Library concentrations were obtained using the Qubit dsDNA HS Assay (ThermoFisher Scientific, Cat. #Q32854). Average library size was determined with the Agilent High Sensitivity DNA Kit (Agilent, Cat. #5067-4626), and accurate library quantification was performed using the Library Quantification Kit - Illumina/Universal Kit (KAPA Biosystems, Cat. #KK4824). Libraries were sequenced on an Illumina NextSeq 500 to generate paired-end 2  151 bp reads.