Computational protocol: Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

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Protocol publication

[…] Sputum and enrichment cultures were thawed to room temperature, and 500 μL of each sample was used for genomic DNA extraction using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA). Purified DNA was submitted to the University of Minnesota Genomics Center (UMGC) for 16S library preparation using a two-step PCR protocol described previously []. A defined mock community was also submitted for sequencing, as were water and reagent controls that did not pass quality control step due to 16S rRNA gene content below detection thresholds. Raw sequence reads were obtained from UMGC and analyzed using a QIIME [] pipeline developed by the UMGC. The average number of reads per sample after filtering and taxonomic assignment was 2.2 x 105, with the minimum and maximum reads per sample of 5.6 x 103 and 5.1 x 105, respectively. Read pairs were stitched together and 16S amplicon primers were removed using PandaSeq (version 2.7)[]. Fastq files were merged and sequence IDs converted to QIIME format using a custom perl script. Chimeric sequences were detected using the QIIME (version 1.8.0) script identify_chimeric_seq.py function, using the usearch61 method. Open reference OTU picking was performed using the pick_open_reference_otus.py script, using the usearch61 method and the Greengenes 13_8 16S rRNA reference database [] clustered at 97% similarity. MetaStats was used to detect differentially abundant features of the mucin-enriched bacterial community []. […]

Pipeline specifications

Software tools QIIME, PANDAseq, USEARCH, Metastats
Databases Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Pseudomonas aeruginosa, Bacteria, Homo sapiens
Diseases Cystic Fibrosis, Pulmonary Disease, Chronic Obstructive
Chemicals Carbon, Fatty Acids