Dear Nicholas Bokulich,
Thanks that does help a lot. I was definitely planning on using quality filter joined prior to deblur and OTU picking. I know there is an option to remove sequences below a particular size in deblur, but is there a way in OTU picking, or does that happen automatically?
Also apparently I was mistaken about my primers, they are still in there. I understand how to trim them using dada 2. I read the Deblur vs DADA2 Questions - #8 by benjjneb thread which indicated this would be a problem in OTU picking and Deblur. Is there currently a way to trim sequences for those two analyses in QIIME2?
I am truly appreciative of all the help. Still working and learning this process as a part of my PhD, no PI here really has experience with 16S sequencing so the Forum is my best way to learn.
qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path '/home/microbiology/Downloads/manifest2.csv' --output-path paired-end-demux.qza --source-format PairedEndFastqManifestPhred33
qiime vsearch join-pairs --i-demultiplexed-seqs paired-end-demux.qza --o-joined-sequences demux-joined.qza
qiime demux summarize --i-data demux-joined.qza --o-visualization demux-joined.qzv
Current Deblur Path
qiime quality-filter q-score-joined --i-demux demux-joined.qza --o-filtered-sequences demux-joined-filtered.qza --o-filter-stats demux-joined-filter-stats.qza
qiime deblur denoise-16S --i-demultiplexed-seqs demux-joined-filtered.qza --p-trim-length 240 --o-representative-sequences 240-rep-seqs.qza --o-table 240-table.qza --p-sample-stats --o-stats deblur-stats.qza
Current 97% OTU Path
qiime quality-filter q-score-joined --i-demux demux-joined.qza --p-min-quality 30 --o-filtered-sequences demux-joined-filtered2.qza --o-filter-stats demux-joined-filter-stats2.qza
qiime vsearch dereplicate-sequences --i-sequences demux-joined-filtered2.qza --o-dereplicated-table otutable.qza --o-dereplicated-sequences oturep-seqs.qza
qiime vsearch cluster-features-de-novo --i-table '/home/microbiology/97OTU/otutable.qza' --i-sequences '/home/microbiology/97OTU/oturep-seqs.qza' --o-clustered-table 97OTU/table-dn-97.qza --o-clustered-sequences rep-seqs-dn-97.qza --p-perc-identity 0.97 --p-threads 8 --verbose
qiime vsearch uchime-denovo --i-table otutable.qza --i-sequences oturep-seqs.qza --output-dir uchime-dn-out
etc... based upon Identifying and filtering chimeric feature sequences with q2-vsearch — QIIME 2 2018.2.0 documentation