Plug in error from cutadapt

[JoseM (Jose Miguel Seoane Redondo)]

Hello guys! I am triying to remove my primers from demultiplex paired end reads obtained from illumina MiSeq prior to run Deblur. First, I imported my data from a folder called Raw (this was ok) which contained foward and reverse fastq files as follows:
(qiime2-2018.6) [jmseoane@tetox Raw]$ time qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path "/pathTo/manifest.csv"
--source-format PairedEndFastqManifestPhred33
--output-path "/pathTo/Raw/demux-paired-end"

Then I tried to trim my primers using cutadapt as follows:

qiime cutadapt trim-paired --i-demultiplexed-sequences demux-paired-end.qza --p-cores 16 --p-front-f CAGCMGCCGCGGTAA --p-front-r GTGCTCCCCCGCCAATTCCT --output-dir trimmed-sequences

and got the following plugin error:

I attach the log file, I am running on fedora on a 64 cores computer. Any suggestion on what did I make wrong?

Thanks a lot in advance for your help!

Best regards,

Jose

qiime2-q2cli-err-txuolw3d.txt (9.8 KB)

[ebolyen (Evan Bolyen)]

Hey @JoseM,

Thanks for the log! Your environment looks like it's 2018.8, but in your log, the software says its from 2018.6:

/home/jmseoane/miniconda2/envs/qiime2-2018.6/lib/python3.5/site-packages/...

What does qiime info say?

Noteably, there was an issue with trim-paired in 2018.6 and we released a patch version for it (2018.6.1):

Incorrect results produced by q2-cutadapt's trim-paired

Summary We recently discovered a bug related to the q2-cutadapt plugin’s trim-paired command that, in certain situations, can produce incorrect results. In particular, the forward and reverse reads are swapped, causing the underlying cutadapt command to apply the forward read trimming parameters on the reverse reads (and vice versa). Impact This issue impacts the following versions of q2-cutadapt: 2017.12 2018.2 2018.4 If you think you may have been impacted please check your provenance at h…

It's possible you've run into this inadvertently. The reads listed in particular seem to be the wrong direction for the file they state they are coming from.

If your 2018.8 environment is accidentally using 2018.6 it's possible that's the reason for the error.

[JoseM (Jose Miguel Seoane Redondo)]

Hi Evan,

Thank you for your fast reply, yesterday I was triying with both enviroments so maybe I messes up. Today after reading your reply I started a fresh session and looks ok :

But the plugin error is still there:

---

Plugin error from cutadapt:

Command '['cutadapt', '--cores', '16', '--error-rate', '0.1', '--times', '1', '--overlap', '3', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-54hp91u4/sample11_20_L0 '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-54hp91u4/sample11_21_L001_R2_001.fastq.gz', '--front', 'CAGCMGCCGCGGTAA', '-G', 'GTGCTCCCCCGCCAATTCCT', '/tmp/qiime2-archive-p-8c5b-2b8b9e96cbe0/data/sample11_20_L001_R1_001.fastq.gz', '/tmp/qiime2-archive-plqml3qa/4172746b-c23a-4704-8c5b-2b8b9e96cbe0/data/sample11_21_L001_R2_001.fastq.gz']' returned

Debug info has been saved to /tmp/qiime2-q2cli-err-qzse6j2n.log

---

I attach the new log just in case it helps! Thanks a lot in advance for any suggestion you may have.

Regards,

Joseqiime2-q2cli-err-qzse6j2n.txt (9.8 KB)

[thermokarst]

Hey there @JoseM!

I pulled this error out of the log you attached:

cutadapt: error: Reads are improperly paired. Read name 'M03099:46:000000000-BBDY9:1:1101:22679:1783 2:N:0:107' in file 1 does not match 'M03099:46:000000000-BBDY9:1:1101:14264:1773 1:N:0:108' in file 2.

This was raised while processing sample11. The error is saying that your forward and reverse reads don’t match (the aren’t “paired” properly). Double-check your files, pre-import to QIIME 2 to make sure you imported what you intended to.

[JoseM (Jose Miguel Seoane Redondo)]

Hi @ebolyen

You were totally right, removing that sample fixed the issue, thanks a lot and sorry, I will check the log more carefully next time!

Jose