I followed the dada2 tutorial on 16S and ITS amplicon metagenomic data from soil samples. However, when trying to assign a taxonomic rank to my ASV table using DECIPHER, I only get "NAs". When using the dada2 native's Bayesian method, I get "NA" or "Mitochondria" (family). For my 16S workflow, I already tried using "strand = both" to inverse the read orientation, however, same result.
For 16S, my workflow was as follows:
- Removing the primers using cutadapt in R, as explained by the dada2 ITS tutorial
- Removing the adapters using trimmomatic
- Following the remaining dada2 tutorial
I also ran the dada2 tutorial on my 16S files without removing the adapters and by removing the primers using dada2 and trunacting left and right. However, same result.
For my ITS files, I followed the dada2 tutorial.
For 16S, my filtering settings are:
out <- filterAndTrim(fnFs, filtFs, fnRs, filtRs, truncLen=c(198, 190), maxN=0, maxEE=c(4, 4), truncQ=2, rm.phix=TRUE, compress=TRUE, multithread=FALSE)
The quality of my 16S files is not great with a score of only 20 starting from 200 bp. The settings above were provided to me using the tool Figaro. My ITS score is generally better.
The sequencing service provider also included a preliminary summary through sending the FastQ files through their Illumina pipeline. In this summary, their are plenty of identified Genus and Species, which makes me believe that the general quality of the FastQ files is okay.
For 16S, I used the Silva 16S reference file for DECIPHER which can be found here: http://www2.decipher.codes/Downloads.html and for ITS the Unite database.
I'm out of ideas on what to do next and would appreciate any help in this matter.