I am running a QIIME2-2021.4 Virtual Box with linux environment on WSL2. I am attempting to duplicate the results of a paper for a larger meta-analysis effort. Their methods are as follows:
Primers, 27F and 534R were used. Sequences were quality trimmed (Q20) and reads shorter than 200 bases were removed. Due to amplicon size and quality trimming, forward and reverse reads could not be consistently merged, thus only the forward read was used for analyses. Subsequently, each sample sequence set was sub-sampled to the smallest sample size to avoid analytical issues associated with variable library size. Sub-sampled data were pooled and renamed and clustered into operational taxonomic units (OTU) at 97% similarity.
When I looked at the distribution of reads, the overall quality looked poor so in DADA2 I decided to --p-trim-left 5 \ and --p-trunc-length 95. I also tried to truncate at 120, 150 and 200 to troubleshoot.
Downstream when I attempted to visualize using qiime taxa barplot, only 2 samples had any sub-classification beyond domain. I also tried to cutadapt to remove non-biological sequences which wasn't fruitful.
Any suggestions on troubleshooting would be welcomed!