title: ea-transcriptomics
design: For intubated patients, RNA was extracted from endotracheal aspirates (EAs). Specimens were deoxyribonuclease (DNase)–treated, and human cytosolic and mitochondrial ribosomal RNA was depleted. Complementary DNA (cDNA) synthesis uses a random hexamer approach to capture human coding and noncoding RNA transcripts, as well as nonhuman RNAs. cDNA libraries were sequenced with paired-end reads at a target depth of 50 million reads per sample using a NovaSeq 6000 sequencer with a S4 flowcells and 200 cycle flow cells. Human reads are aligned to the GRCh38 reference genome and quality-controlled by total counts per library and median coefficient of variation (CV) coverage. Raw counts for these genes are normalized across libraries according to the “Trimmed Means of M Values” (TMM) method, as implemented in the edgeR package for downstream analysis. Remaining nonhuman reads are aligned using the National Center for Biotechnology Information (NCBI) nucleotide and nonredundant protein databases followed by assembly of the reads matching each taxon detected. Repeated control and participant samples are sequenced within each sequencing batch to mitigate batch effects throughout the study.