Apologies - I found past discussions on this topic but the topics were closed for discussion and the suggestions did not work for me, so I am hoping someone can help to find a solution. We had a fantastic QIIME2 workshop in Melbourne 2 weeks ago and I was finally able to give it a try last week with 3 samples and then a larger sample set and mostly it seemed to work okay. However, after I ran dada2 using the default settings the number of sequences reduced dramatically, and I ended up with only about 25-30% of the original reads. I tried to run again not trimming further the F/R sequences (as per below) but it didn’t change the results. I have added some of the files including unprocessed F and R sequences for the samples, the demux.qzv (quality) and denoised.qzv (number of reads) files at https://drive.google.com/open?id=1-xn8MwEu9-pUlPRuKSc87yhpwUkvstom.
Briefly, I follow the EMP protocol to amplify V4-V5 region in the MiSeq (fragment is ~450 bp, sequencing 2x300 bp), so I have forward and reverse sequences that need to be joined.
I am using QIIME2 v11.18 installed in a server and I used the following commands:
To import: qiime tools import --input-path ~/xxxxx --output-path demux.qza --input-format CasavaOneEightSingleLanePerSampleDirFmt --type ‘SampleData[PairedEndSequencesWithQuality]’
To summarise demultiplexing results: qiime demux summarize --i-data demux.qza --o-visualization demux.qzv
To join F/R: qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trunc-len-f 0 --p-trunc-len-r 0 --o-representative-sequences rep-seqs2.qza --o-table table2.qza --o-denoising-stats denoised2.qza
This was followed by metadata tabulate and feature table.
Based on my experience with QIIME 1 and based on what others have written this is not expected. I was wondering if there is anything else I can try to do to reduce the waste of reads?
This has happened now with 2 datasets that were produced by different MiSeq runs so I don’t think it is an issue with the run itself either (and the protocol is exactly the one in the EMP website).
Thank you so much! Any help will be much appreciated!