Computational protocol: Sputum Bacterial and Fungal Dynamics during Exacerbations of Severe COPD

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Protocol publication

[…] The V4 hyper-variable region was sequenced by overlapping using the PE250 sequencing strategy. For quality-control purposes, we removed all of the sequences that contained ambiguous reads (N) and any sequences that contained mismatches in the forward or reverse primers. These clean, non-continuous sequences were then screened for chimeras using UCHIME []. A total of 3034133 high-quality sequencing reads of the 16S rRNA gene were generated after UCHIME use, 517850 of which belonged to fungi. The subsequent analyses were implemented using QIIME (1.5.0). Each 16S rRNA gene sample was normalized to 22000 sequences, and we used the non-normalized data to perform later fungal analyses. Before we analyzed the fungal data, we transformed the data using the constituent ratio. The sequences were then clustered using UCLUST, with a threshold distance set to 0.03, which corresponded to the species level. The Ribosome Database project (RDP) algorithm was applied to classify the representative sequences of each operational taxonomic unit (OTU) [], the Shannon and PD_whole_tree indices were used to analyze alpha diversity, and PCoA analysis using QIIME based on the UniFrac distance was implemented []. The sequences were classified at the genus level and grouped into the next lowest taxonomic level. The sequences that were clearly fungal were used in an additional BLAST [] analysis using the NCBI databank. Beta-diversity analyses were implemented based on the UniFrac distance, and all statistical analyses were performed using SigmaPlot 12.0. […]

Pipeline specifications

Software tools UCHIME, QIIME, UCLUST, SigmaPlot
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Bacteria, Fungi, Homo sapiens
Diseases Acinetobacter Infections, Pulmonary Disease, Chronic Obstructive