Denoising with Deblur
Deblur is an alternative method to produce a set of denoised sequences. While DADA2 can natively support paired-end reads, Deblur can only manage single-end reads.
We can merge the paired end reads using VSEARCH, that is available as a Qiime2 plugin
Merge reads with VSEARCH
The complete documentation of the q2-vsearch plugin contains all the available parameters.
This subprogram merge the overlapping pairs to produce a set of single-end FASTQ file, and will work only when the amplicon are designed with the overlap.
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qiime vsearch join-pairs \
--i-demultiplexed-seqs raw-reads.qza \
--o-joined-sequences joined-reads.qza \
--p-threads 8
Sequence quality control and feature table construction
This method filters sequence based on quality scores and the presence of ambiguous base calls.
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qiime quality-filter q-score \
--i-demux joined-reads.qza \
--o-filtered-sequences joined-filtered.qza \
--o-filter-stats joined-filter-stats.qza
Denoising with deblur
Deblur requires to process a set of single end reads truncated at the same length (which now should be decided after)
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qiime deblur denoise-16S \
--i-demultiplexed-seqs joined-filtered.qza \
--p-trim-length 245 \
--p-sample-stats \
--p-jobs-to-start 32 \
--o-stats deblur-stats.qza \
--o-representative-sequences rep-seqs-deblur.qza \
--o-table table-deblur.qza
As usual, we can produce visualization artifacts to summarize the content of:
- representative sequences
- denoising statistics
- feature table
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qiime deblur visualize-stats \
--i-deblur-stats deblur-stats.qza \
--o-visualization deblur-stats.qzv
qiime feature-table tabulate-seqs \
--i-data rep-seqs-deblur.qza \
--o-visualization rep-seqs-deblur.qzv
qiime feature-table summarize \
--i-table table-deblur.qza \
--m-sample-metadata-file metadata.tsv \
--o-visualization table-deblur.qzv