# There are small unmatched reads in paired reads of kneaddata result

**URL:** <https://forum.biobakery.org/t/there-are-small-unmatched-reads-in-paired-reads-of-kneaddata-result/7731>\
**Category:** KneadData\
**Created:** [January 9, 2025, 2:19pm UTC](https://forum.biobakery.org/t/there-are-small-unmatched-reads-in-paired-reads-of-kneaddata-result/7731 "2025-01-09T14:19:10Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![Kirby](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/kirby/32/1600_2.png) [@Kirby](https://forum.biobakery.org/u/Kirby)\
**Post date:** [January 9, 2025, 2:19pm UTC](https://forum.biobakery.org/t/there-are-small-unmatched-reads-in-paired-reads-of-kneaddata-result/7731/1 "2025-01-09T14:19:10Z")

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Dear bioBakery team,

I am using the kneaddata-v0.12.0 from bioconda for QC of my metagenomic raw data.

To date, kneaddata has worked perfectly for all my purposes.

However, when I recently tried profiling my QCed data using mOTUs4, I got an error for `[kneaddata_result]_paired_1.fastq` and `[kneaddata_result]_paired_2. fastq`, indicating that they are not paired.

This error is due to a mismatch in the header between forward and reverse reads.  
This problem did not occur with MetaPhlAn and Kraken, which I have been using for a long time.

In my case, I have compromised to proceed with the next step of mOTUs by removing the inconsistent reads. Since there were only 88 unmatched reads in each forward and reverse reads, this is a very small number compared to the total of 31,276,232 reads.

I assume that this is because bowtie2 runs in unpaired mode within kneaddata, but it is only an guess.

I believe that it would be not a significant issue for bowtie2 to run in unpaired mode since the size of my data is large enough, but I am unsure why such a small number of reads are avoided in the process of matching pairs.

In this case, the number of unpaired reads was minimal, but I am concerned that the number might be large to ignore in other cases of my data.

I would be grateful if you could give me your opinion on this.

I have attached the kneaddata log of the data I used and unique headers detected when tried running mOTUs4.

[stool\_SG\_001\_1\_kneaddata.log.txt](https://forum.biobakery.org/uploads/short-url/1ydQRY0whN6tosP5OgERdh115Vd.txt) (28.2 KB)  
[unique\_headers\_detected.txt](https://forum.biobakery.org/uploads/short-url/yG0mEEtAF3q4TsArPudRk9PVA5S.txt) (6.7 KB)

Best regards,  
Kirby

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<div class="post-metadata">

**Author:** ![Kirby](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/kirby/32/1600_2.png) [@Kirby](https://forum.biobakery.org/u/Kirby)\
**Post date:** [January 14, 2025, 9:02am UTC](https://forum.biobakery.org/t/there-are-small-unmatched-reads-in-paired-reads-of-kneaddata-result/7731/2 "2025-01-14T09:02:03Z")

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I found that this problem was caused by mOTUs4 not kneaddata.  
I checked for “differing read headers” that mOTUs4 pointed to for kneaddata results by manually and using BBMap.  
I could not find any mismatched headers.

I have flagged this post to the admin.

Thanks,  
Kirby