I am new in QIIME2 and I am working with paired end reads that came direct from Illumina sequencing. So I have three archives that I imported with the command:
qiime tools import --type EMPPairedEndSequences --input-path paired-end-sequences --output-path paired-end-sequences.qza
And then I demultiplexed with:
qiime demux emp-paired --m-barcodes-file cintiamapping4.tsv --m-barcodes-column BarcodeSequence --i-seqs paired-end-sequences.qza --o-per-sample-sequences demux_novo.qza --p-rev-comp-barcodes
When I look my demux_novo.qzv data I find high sequence count on my sample (illustrated bellow) and the following quality plot:
After I run denoise with DADA2 the number of sequence count is really low!
qiime dada2 denoise-paired --i-demultiplexed-seqs demux_novo.qza --p-trim-left-f 100 --p-trim-left-r 100 --p-trunc-len-f 260 --p-trunc-len-r 260 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats denoising-stats.qza
I tried trim and trunc with several combination… but nothing gets better than the number I get with trim 100 and trunk 260. The following table.qzv illustrate what is the best I got so far…
table.qzv (320.4 KB)
I also tried use Deblur but I also get a low sequence count… actually I get no sequence.
Should I run using alternative methods? I was starting using this tutorial (https://docs.qiime2.org/2018.8/tutorials/otu-clustering/) with my seqs.fna from QIIME1 and filtering with https://docs.qiime2.org/2018.8/tutorials/chimera/
What should I do? Any help, tips, suggestions would be very appreciated!
Happy Holidays to all!
And many thanks in advance!