I am totally new user of qiime2 and don’t have experience in using Qiime1.
Recently I start to use QIIME2(2019.10) to do 16S analysis. And I met a problem regarding filtering reads I guess.
I used conda to install it. (as describing in this linkage about linux system:https://docs.qiime2.org/2019.10/install/native/#install-qiime-2-within-a-conda-environment)
Import command:import pair-end reads(clean data)
qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path se-64-manifest \
--output-path /sbidata/lzhang/201911_hydractinia/RawData/16S_data/output/demux.qza \
Description of my sample after import into qiime2
I totally have 73 samples before doing denoising
Qualtiy as belows
Command as follows: denoise and visualize step
based on the qulity plots, i set the trim length 300, which means I don’t want to trim reads. Cause it seems the reads have good quality. Right?
qiime dada2 denoise-paired \ --i-demultiplexed-seqs demux.qza \ --p-trunc-len-f 300 \ --p-trunc-len-r 300 \ --o-table table.qza \ --o-representative-sequences rep-seqs.qza \ --o-denoising-stats denoising-stats.qza qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file 201912_hydractinia_metadatafile_Qiime2.tsv
Then I used qiime tools view to check the results
I got only one sample left …
stats of this procedure
It seems that other samples don’t pass the filter parameters?
So I check the parameter of overlap, the default overlap is 20.
In order to check if this is the problem. I rerun the denoise procedure by setting 0.
qiime dada2 denoise-paired \ --i-demultiplexed-seqs /sbidata/lzhang/201911_hydractinia/RawData/16S_data/output/demux.qza \ --p-trunc-len-f 0 \ --p-trunc-len-r 0 \ --o-table table.qza \ --o-representative-sequences rep-seqs.qza \ --o-denoising-stats denoising-stats.qza
And this time, I got results of total 77 samples in table.
I have a few questions
I have read some questions relating this, but it seems that no results are like mine…only 1 sample left.
Q1. I am not sure if I set the parameter to 0, then the quality of the result will be low? Can I set it to 0? And will affect the results? If it affects results, do you have any suggestions for me?
Q2. I also have merged reads which are results from the sequencing company. Do I need to analyse directly with the merged reads?
Q3. Should I using deblur instead and try again?
Trully thanks for your help!