Nephele runs the DADA2 R package v1.18 following the steps in the package authors' Big Data workflow including optional use of DECIPHER package v2.18. We make some minor modifications of the parameters used. Additionally, we construct a phylogenetic tree using QIIME 2 v2022.2. Our pipeline is outlined below. If you are new to DADA2, it might be helpful to read through the DADA2 Tutorial.
trim leftparameter be increased by 15 bp (on top of any primer lengths). This option is in beta, and has not been extensively tested. If you have Ion Torrent data, we are interested in your feedback - please email us!
For paired-end data only.
IDTAXA. (Default: rdp)
IDTAXAwill use its own SILVA database. See Databases below.
IDTAXA, we use the authors' modified SILVA v132/v138 SSU trained classifier. More information in the DECIPHER FAQ.
This is the pipeline workflow along with the outputs given at each step. We link to the specific DADA2 R functions that are used.
Preprocess sequence data with filterAndTrim. The maximum expected errors (maxEE), trim left (trimLeft), truncation quality score (truncQ), and truncation length (truncLen) parameters can be set by user options. The filtered sequence files, *_trim.fastq.gz, are output to the filtered_data directory.
Learn the error rates with learnErrors. The error rate graphs made with plotErrors are saved as errorRate_R1.pdf, errorRate_R2.pdf. The error profiles,
err, are also saved as a list R binary object in the intermediate_files directory.
For paired-end data, merge the overlapping denoised reads with mergePairs. The default minimum read overlap, minOverlap, parameter is set to 12. Trim overhanging sequence (trimOverhang), just concatenate (justConcatenate), and maximum mismatches (maxMismatch) can be set as user options. The sequence table,
seqtab, containing the final amplicon sequence variants (ASVs), is saved as an R binary object (seqtab.rds) to the intermediate_files directory.
Filter out ASVs of length less than 75 bp. The resulting sequence table is saved as seqtab_min75.rds. Also, filter out chimeras with removeBimeraDenovo, if the option is chosen, and save that result as seqtab_nochimera.rds in the intermediate_files folder.
Depending on the user options for taxonomic assignment and reference database, classify the remaining ASVs taxonomically with
rdp using assignTaxonomy (default). The minBoot parameter for minimum bootstrap confidence is set to 80 and tryRC is set to TRUE, so the best match from each sequence or its reverse-complement is used. Add species annotation to the taxonomic identification using addSpecies. This final result is saved as a biom file taxa.biom.
IDTAXA using IdTaxa from the DECIPHER R package. The final result will be saved as taxa.biom
The final results are also saved as a tab-separated text file OTU_table.txt. The final sequence variants used for taxonomic classification are output as seq.fasta. A summary of the counts in the OTU table is saved to otu_summary_table.txt.
Construct a phylogenetic tree from ASVs using QIIME 2 align-to-tree-mafft-fasttree pipeline with default parameters. This produces tree files in Newick format in the phylo directory: unrooted_tree.nwk and rooted_tree.nwk.
See Pipeline Steps above for more details on how these files were made.
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McMurdie PJ and Paulson JN (2016). biomformat: An interface package for the BIOM file format. https://github.com/joey711/biomformat/.
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Escapa, I. F., Chen, T., Huang, Y., Gajare, P., Dewhirst, F. E., and Lemon, K. P. (2018). "New Insights into Human Nostril Microbiome from the Expanded Human Oral Microbiome Database (eHOMD): a Resource for the Microbiome of the Human Aerodigestive Tract." MSystems, 3(6), e00187-18. doi: 10.1128/mSystems.00187-18.
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Bolyen, E. et al. (2019) "Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2." Nature Biotechnology, 37(8), 852–857. doi: 10.1038/s41587-019-0209-9.