Dear developers and community,
Thank you for your help, and for supporting Qiime:
Problem Description
Problem: I am yielding little to know sequences when merging during de-noising step with the following call to qiime dada2 denoise-paired (as per below).
Methods: I have at hand 18S sequences. The library length distribution per Bioanalyser has its mode about 481 bp. I have sequenced 2x250 bp on a MiSeq version 2 kit. FasQC check of all fastq files revealed nominal sequencing performance. I am receiving demultiplexed fastq files from the sequencing facility. Library design follows http://www.earthmicrobiome.org/protocols-and-standards/18s/. I am guessing the overlap is too small, and I would have had better performance if I used the 2x300 kit.
Results: qiime tools view on the summary stats reveal after de-noising and merging for a typical sample, that most reads can’t be merged. See below - the best result I ever get is 10% of input reads merged, trimming parameter adjustment doesn’t help.
- input: 175777
- filtered: 128898
- denoised: 128898
- merged: 24
- non-chimeric: 24
Could you help me?:
- How can I improve merging?
- can I adjust the overlap setting (to less then 20 bp?)
- Less stringent filtering during the earlier
importandcuadaptsteps I do?
- How can I analyse my resulst if merging continues to be unsucessful?
- Perhaps by analysing only the forward reads?
- Which tool / plugin would I use?
Any comment would be appreciated. Thank you!
Code Snippet
# define input locations
# ---------------------------------
inpth[1]='Zenodo/Qiime/030_18S_10410623-trimmed.qza'
# define output locations
# ---------------------------------
otpth_seq[1]='Zenodo/Qiime/050_18S_10410623-seq.qza'
otpth_tab[1]='Zenodo/Qiime/050_18S_10410623-tab.qza'
otpth_stat[1]='Zenodo/Qiime/050_18S_10410623-stat.qza'
# trimming parameters 18S - aiming for Phred 20
# ---------------------------------------------
lenf[1]='50'
lenr[1]='50'
# run script
# ----------
for ((i=1;i<=1;i++)); do
qiime dada2 denoise-paired \
--i-demultiplexed-seqs "$trpth"/"${inpth[$i]}" \
--p-trunc-len-f "${lenf[$i]}" \
--p-trunc-len-r "${lenr[$i]}" \
--p-n-threads "$thrds" \
--o-representative-sequences "$trpth"/"${otpth_seq[$i]}" \
--o-denoising-stats "$trpth"/"${otpth_stat[$i]}" \
--o-table "$trpth"/"${otpth_tab[$i]}"
done
Latest used Qiime version
qiime info
System versions
Python version: 3.5.5
QIIME 2 release: 2018.11
QIIME 2 version: 2018.11.0
q2cli version: 2018.11.0
Installed plugins
alignment: 2018.11.0
composition: 2018.11.0
cutadapt: 2018.11.0
dada2: 2018.11.0
deblur: 2018.11.0
demux: 2018.11.0
diversity: 2018.11.0
emperor: 2018.11.0
feature-classifier: 2018.11.0
feature-table: 2018.11.0
fragment-insertion: 2018.11.0
gneiss: 2018.11.0
longitudinal: 2018.11.0
metadata: 2018.11.0
phylogeny: 2018.11.0
quality-control: 2018.11.0
quality-filter: 2018.11.0
sample-classifier: 2018.11.0
taxa: 2018.11.0
types: 2018.11.0
vsearch: 2018.11.0
Application config directory
/Users/paul/Library/Application Support/q2cli
Getting help
To get help with QIIME 2, visit https://qiime2.org