Metagenomic Sequencing of Biological Samples

Specimens underwent RNA extraction and metagenomic sequencing, as previously described^{30 (link)}. Briefly, RNA was extracted from 200 μL of specimen in DNA/RNA shield using bead-based lysis and the Zymo Pathogen Magbead kit (Zymo). We also processed negative control samples (water and HeLa cell RNA) to account for background contamination. All samples were spiked with RNA standards from the External RNA Controls Consortium (ERCC)^{44 (link)}. Samples were DNase treated, depleted of cytosolic and mitochondrial rRNA using FastSelect (Qiagen, Germantown, MD), and reverse transcribed to generate cDNA. Sequencing libraries were constructed using the NEBNext Ultra II Library Prep Kit (New England Biolabs, Ipswich, MA). Libraries underwent 146 nucleotide paired-end sequencing on an Illumina Novaseq 6000 instrument.