Computational protocol: Microbial aerosol liberation from soiled textiles isolated during routine residuals handling in a modern health care setting

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Protocol publication

[…] Airborne bacterial profiles were determined by broad-range amplification on an Illumina MiSeq platform to sequence 16S ribosomal ribonucleic acid (rRNA) amplicons; these were generated using broad-range PCR primers that circumscribe approximately a 300 bp of the variable V1V2 variable region of []. PCR products were normalized using a SequalPrep™ kit (Invitrogen, Carlsbad, CA), pooled, lyophilized, purified, and concentrated using a DNA Clean and Concentrator Kit (Zymo, Irvine, CA). Pooled amplicons were quantified using Qubit Fluorometer 2.0 (Invitrogen, Carlsbad, CA), and each pool was diluted to 4 nM and denatured with 0.2 N NaOH at room temperature. The denatured DNA was diluted to 15 pM and augmented with 25 % of PhiX DNA (manufacturer’s control) immediately prior to sequencing. Paired-end sequencing was performed with version 2.0 of the Miseq Control Software, using a 500-cycle reagent kit (v 2.0). The resulting sequences were sorted by barcodes in the paired reads with a python script and assembled using PHRAP [, ]. These assemblages were trimmed over a moving window of five nucleotides until their average quality met or exceeded 20. Pairs that did not assemble, or trimmed sequences with more than 1 ambiguity (any non-specific base call) or shorter than 200 bp, were discarded. Potential chimeras were identified with UCHIME (usearch6.0.203_i86linux32) [] using the Schloss SILVA reference sequences [] and removed from subsequent analyses. Assembled sequences were aligned and classified with SINA 1.2.11 [] using the conglomerate of bacterial sequences in SILVA 115NR99 []. Assembled sequences were aligned and classified with SINA (1.2.11) using the 629,124 bacterial sequences as reference configured to yield the SILVA taxonomy. […]

Pipeline specifications

Software tools UCHIME, USEARCH
Application 16S rRNA-seq analysis
Organisms Homo sapiens