Workflow of the wet laboratory, bioinformatics, and data filtering procedures in the process of data filtering for 16S rRNA amplicon sequencing. Workflow.

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Workflow of the wet laboratory, bioinformatics, and data filtering procedures in the process of data filtering for 16S rRNA amplicon sequencing. Workflow of the wet laboratory, bioinformatics, and data filtering procedures in the process of data filtering for 16S rRNA amplicon sequencing. Reagent contaminants were detected by analyzing the sequences in the NCext and NCPCR controls. Sequence number thresholds for correcting for cross-contamination (TCC) are OTU and run dependent and were estimated by analyzing the sequences in the NCmus, NCext, NCPCR, and PCindex controls. Sequence number thresholds for correcting for false-index-pairing (TFA) values are OTU and run dependent and were estimated by analyzing the sequences in the NCindex and PCalien controls. A result was considered positive if the number of sequences was >TCC and >TFA. Samples were considered positive if a positive result was obtained for both PCR replicates. *, see Kozich et al. (18) for details on the sequencing. Maxime Galan et al. mSystems 2016; doi:10.1128/mSystems.00032-16