The forward and reverse sequences do not match after importing data

Dear all!
I'm having issues with denoising after importing data normally using QIIME2.

Plugin error from dada2:

An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

Debug info has been saved to /tmp/qiime2-q2cli-err-as918jc4.log
The error is that "Error in (function (fn, fout, maxN = c(0, 0), truncQ = c(2, 2), truncLen = c(0, :
Mismatched forward and reverse sequence files: 86106, 63444."
So I return to the "demux.qzv" file,found that the problem does exist.

My import order is
time qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path mainfest.txt --output-path demux.qza --input-format PairedEndFastqManifestPhred33
My question is why this problem occurs and how can i fix it.

I would appreciate any help![question|690x130]

Hello @Agatsuma,

Welcome to the forums! :qiime2:

You have correctly diagnosed the error: some reads are missing from your files such that the number of forward and reverse reads do not match.

This is usually because the reads were pre-filtered (maybe by the sequencing core?) before they were imported using the command you mentioned. Qiime2 works best with raw data, so if you have access to the unprocessed data, that's what I would try next.

Do you still have access to the raw data directly off the sequencer?

Thanks for your reply! :smiley:

My data is rawdata in my opinion,but I cant prove it. Because the data was returened form the sequencing company after I send my samples to the company.

So did you mean that the rawdata I get from the company may not the really rawdata?

Besides,the paired sequencing data size I get are the same.1
I dont know why the number of reads imported will vary when the file size is the same.

I would appreciate for your reply!

Interesting! :thinking:

Well, the Illumina platform always makes the same number of forward and reverse reads in the same order, inside the fastq files. If reads are missing, like they are here, I suspect some filtering has been done.

Or maybe I'm wrong!

May I ask what sequencing company you used?

1 Like

I'm so sorry for not replying to you for a long time.I have figured out the reason why the error appear.The reason is that I misspelled a number of manifest.txt.

I'm so sorry for westing your time again!


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