Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

MS Zinter; CC Dvorak; MY Mayday; K Iwanaga; NP Ly; ME McGarry; GD Church; LE Faricy; CM Rowan; JR Hume; ME Steiner; ED Crawford; C Langelier; K Kalantar; ED Chow; S Miller; K Shimano; A Melton; GA Yanik; A Sapru; JL DeRisi

To avoid spurious alignments and sequencing artifacts, we only included read pairs where both ends aligned to the same taxonomic ID. The prevalence of each microbe was described (1) relative to other microbes in the same sample, wherein we normalized sequencing reads per million total sequencing reads (rpm); and (2) relative to the same microbe in other samples in the cohort, wherein we normalized sequencing reads as the number of standard deviations above or below the mean log10-transformed rpm for the total cohort (Z-score) (23) . Outliers were defined as microbes with RNA alignments that were both abundant within a sample (≥10% of all bacterial reads in a sample, or >1 rpm for fungi and viruses) and ≥2 standard deviations above the cohort mean for that particular microbe (Z-score ≥2). A subset of outliers were confirmed via orthogonal assays. The Simpson's Diversity Index was used to associate the loss of bacterial diversity with the presence of outlier microbes (detailed methods in Supplemental Text 5) (24) .