Hello!
I sent my DNA samples off to a sequencing center for sequencing and had some basic analyses performed by one of their bioinformaticians. I'm trying to walk through what he did so I can understand the code myself, but have run into some issues.
Qiime version: qiime2-2023.5 (installed in a conda environment, and I'm working in Terminal)
The code I've run so far is as follows:
qiime tools import \
--type "SampleData[SequencesWithQuality]" \
--input-format SingleEndFastqManifestPhred33V2 \
--input-path ./manifest.tsv \
--output-path ./demux_seqs_original.qza
qiime cutadapt trim-single --i-demultiplexed-sequences demux_seqs_original.qza --p-cores 8 --p-minimum-length 240 --o-trimmed-sequences demux_seqs.qza --p-adapter GTGYCAGCMGCCGCGGTAA...ATTAGAWACCCBNGTAGTCC --p-discard-untrimmed
qiime demux summarize --i-data ./demux_seqs.qza --o-visualization ./demux_seqs.qzv
My manifest.tsv file looks like, but for brevity, I'm just listing the first 2:
|sample-id|absolute-filepath|
|M01|/home/<username>/scratch/rawreads_16S/samples/1691-01-M1_S291_L001_R1_001.fastq.gz|
|M02|/home/<username>/scratch/rawreads_16S/samples/1691-02-M2_S292_L001_R1_001.fastq.gz|
demux_seqs.qzv
What else should I try?
timanix
(Timur Yergaliyev)
September 22, 2023, 7:48am
2
Hello!
I would reorder and separate commands:
First, import files:
qiime tools import \
--type "SampleData[SequencesWithQuality]" \
--input-format SingleEndFastqManifestPhred33V2 \
--input-path ./manifest.tsv \
--output-path ./demux_seqs_original.qza
Then create a visualization to check the data:
qiime demux summarize \
--i-data ./demux_seqs_original.qza \
--o-visualization ./demux_seqs_original.qzv
And only after checking sequences info run cutadapt:
qiime cutadapt trim-single \
--i-demultiplexed-sequences demux_seqs_original.qza \
--p-cores 8 \
--p-minimum-length 240 \
--o-trimmed-sequences demux_seqs.qza \
--p-adapter GTGYCAGCMGCCGCGGTAA...ATTAGAWACCCBNGTAGTCC \
--p-discard-untrimmed
qiime demux summarize \
--i-data ./demux_seqs.qza \
--o-visualization ./demux_seqs.qzv
If sequences are Ok before cutadapt, I would play with --p-minimum-length
and double check --p-adapter
since --p-discard-untrimmed
option is trashing any sequence with no adapter found in it.
1 Like
Thank you so much!
Here is a visualization of the demux_seqs_original.qzv
The minimum scores are mainly influenced by my NTCs.
And here are screenshots of the interactive quality plot:
Based on this, would you recommend making --p-minimum-length 154 in cutadapt instead?
timanix
(Timur Yergaliyev)
September 23, 2023, 7:59am
4
I would disable it at all or set to lower value (150) to get rid of sequences shorter than threshold.
system
(system)
Closed
October 24, 2023, 1:59pm
5
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