Wishing all a very happy 2020.
I have a problem with the Qiime2 analysis. I have done my data analysis in different ways using Qiime2 commands. In my first analysis, I have used cutadapt to trim my raw sequences and then did dada2 denoise using the following parameters:
qiime cutadapt trim-paired --i-demultiplexed-sequences bac-paired-end-demux.qza --p-cores 28 --p-front-f CCTACGGGNBGCASCAG --p-anywhere-f AGATCGGAAGAG TGGAATTCTCGG GATCGTCGGACT CTGTCTCTTATA CGCCTTGGCCGT --p-front-r GACTACNVGGGTATCTAATCC --p-anywhere-f AGATCGGAAGAG TGGAATTCTCGG GATCGTCGGACT CTGTCTCTTATA CGCCTTGGCCGT --o-trimmed-sequences trim-bac-paired-end-demux.qza --verbose qiime dada2 denoise-paired --i-demultiplexed-seqs trim-bac-paired-end-demux.qza --p-trunc-len-r 0 --p-trunc-len-f 0 --p-max-ee-f 4.0 --p-max-ee-r 4.0 --p-trunc-q 2 --p-n-threads 0 --o-table trim-5-paired-table.qza --o-representative-sequences trim-5-rep-seqs-paired.qza --o-denoising-stats trim-5-denoising-stats-paired.qza --p-chimera-method consensus --verbose
After the Open reference OTU picking using Green gene database, chimera removal using qiime vsearch uchime-denovo command, taxonomic classification using qiime feature classifier classify-sklearn and filtering the mitochondrial and chloroplast sequences using qiime taxa filter-table and qiime taxa filter-seqs, we obtained the following final table:
table-nc-wobl-no-mitochondria-no-chloroplast.qzv (2.5 MB)
The final feature table with the taxonomy information was obtained by the qiime tools export, biom add metadata and biom convert commands. I am attaching the feature table with taxonomy.tsv file.
feature-table-with-taxonomy.tsv (686.9 KB)
But when I checked the OTU information in the feature table, there is a dominant number of OTUs assigned only up to bacteria_ level. They even account for the top 10 bacterial taxa for most of my samples.
I don’t know how to solve this problem. Also when I copied the respective OTU sequences and compared in Ezbiocloud database the sequence was getting classified upto species level and above 99% similarity was shown to typestrain sequences deposited in the database.
But the sequence orientation was found to be reverse in the result.
I am wondering whether the sequence orientation has something to do with OTU picking and taxonomic assignment. Is there any steps to check the raw forward and reverse read orientation and solving this issue.
Sorry for such a long query. Looking forward for solutions as I am stuck and confused at this stage.