Nebnext ultra 2 direction rna sequencing prep kit

Metagenomic and metatranscriptomic sequencing libraries were prepared as described in ref. ^{2 (link)} Cabral 2020. For metagenomic libraries 100 ng of DNA was used with the NEBNext® Ultra II FS DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) as per manufacturer’s instructions to generate a pool of fragments at 300 bp ± 50 bp. Metatranscriptomic libraries were created with total RNA (1 μg) using the MICROBExpress kit (Invitrogen, Carlsbad, CA, USA), NEBNext® rRNA Depletion Kit for Human/Mouse/Rat (New England BioLabs, Ipswich, MA, USA), and the NEBNext® Ultra II Direction RNA Sequencing Prep Kit as per the manufacturers’ instructions to generate a pool of fragments at 300 bp ± 50 bp. Metagenomic and metatranscriptomic libraries were pair-end sequenced (2 × 150 bp) on the Illumina HiSeq X Ten. An average of 12,928,385 reads per metagenomic sample and 52,848,755 reads per metatranscriptomic sample. A control metagenomic sequencing library was made using the Zymobiomics Microbial Community Standard (D6300, Zymo Research) (Irvine, CA United States) and added to the sequencing run. This data was used to validate the accuracy of the sequencing run. Sequencing of this standard resulted in relative abundances near the theoretical composition with all community members identified.