Sample prep comparison for wastewater sequencing
This metatranscriptomic library was generated from the filtrate fraction of a wastewater sample filtered through a 0.45-m membrane, replicate 1. The concentration of the total RNA was obtained using the Qubit RNA HS Assay, and quality was determined using the Agilent Bioanalyzer RNA Pico Assay. Ribosomal RNA was depleted from total RNA using the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, Cat. #20037135). RNA was converted to cDNA, and adapters and indices were added onto the ends of the fragments to generate Illumina libraries. Libraries were eluted in DNA Elution Buffer (Zymo Research, Cat. #D3004-4-10). The concentration and average size of the libraries were obtained using the TapeStation DNA 5000 Assay Reagents and Screen Tapes (Agilent, Cat. #5067-5589 and #5067-5588). Libraries were normalized and pooled based on TapeStation concentrations, and accurate library quantification of the pool was performed using the Library Quantification Kit - Illumina/Universal Kit (KAPA Biosystems, Cat. #KK4824). The library pool was sequenced using the NextSeq 2000 P3 Reagent Kit (300 cycles) (Illumina) to generate paired-end 2  151 bp reads.