# Barcode Errors Impair Demultiplexing with Cutadapt?
**URL:** https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336
**Category:** User Support
**Tags:** demux, cutadapt
**Created:** [July 29, 2021, 4:41pm UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336 "2021-07-29T16:41:14Z")
**Posts on this page:** 6
**Page:** 1
### Author:  [@Stephan\_Bitterwolf](https://forum.qiime2.org/u/Stephan_Bitterwolf)
#### Post date: [July 29, 2021, 4:41pm UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/1 "2021-07-29T16:41:14Z")
I hope this question can help others working with paired-end sequences that have forward.fastq.gz, reverse.fastq.gz, and a metadata table containing a column with barcodes. My main problem is that the cutadapt demultiplexing identifies very sequences per sample. I believe this is due to a barcode issue.
My methods:
To **import the data** into qiime2 I followed this [qiime2 tutorial](https://docs.qiime2.org/2021.4/tutorials/importing/#multiplexed-paired-end-fastq-with-barcodes-in-sequence).
```
qiime tools import \
--type MultiplexedPairedEndBarcodeInSequence \
--input-path sequences \
--output-path multiplexed-seqs.qza
```
To **demultiplex the data** I used the cutadapt command:
```
qiime cutadapt demux-paired \
--i-seqs multiplexed-seqs.qza \
--m-forward-barcodes-file metadata.tsv \
--m-forward-barcodes-column barcode-sequence \
--p-error-rate 0.1 \
--output-dir demultiplexed_data
```
To **summarize the data** I followed the basic code:
```
qiime demux summarize \
--i-data demultiplexed_data/per_sample_sequences.qza \
--o-visualization demultiplexed_data.qzv
```
**The problem** I encountered was that almost no reads were found for some samples.
I **used a simple grep search** of the unzipped forward reads (`gunzip forward.fastq.gz`) and found that there were _a lot_ of errors in the barcodes themselves. For example, all barcodes started with "TAAGGCGA" followed by 8 unique nucleotides. I used the grep command `grep -o TAAGGCGA........ forward.fastq | sort | uniq` and identified 163 unique variations of the barcodes. **None** of these extracted barcodes exactly **matched the barcodes I supplied** in my metadata.tsv file.
I **increased the number of sequences identified** for each sample by allowing _more errors_ in the barcode matching with the following command:
```
qiime cutadapt demux-paired \
--i-seqs multiplexed-seqs.qza \
--m-forward-barcodes-file metadata.tsv \
--m-forward-barcodes-column barcode-sequence \
**--p-error-rate 0.2** \
--output-dir demultiplexed_data_error.2
```
This identified more sequences. However, I am left feeling unsure about the demultiplexing process.
**Questions** :
Am I right in assuming that my main issue stems from the barcodes?
Is it normal to not have a perfect match for the barcodes?
Or is this user error?
I am using qiime2-2021.4, on a windows 10 computer using the windows subsystem for Linux. I have used this computer to analyze microbiome data previously so I don't think it is my hardware.
Thank you for your time and support.
---
### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [August 2, 2021, 7:24pm UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/2 "2021-08-02T19:24:35Z")
Hi @Stephan_Bitterwolf, thanks for reaching out!
Based on the test you ran by increasing the error rate, my assumption here would be that the main issue is caused by your barcodes - but I will tag a couple of folks on this to see what additional insight we can provide here. Thanks for your patience!
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 3, 2021, 2:33pm UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/5 "2021-08-03T14:33:02Z")
---
### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [August 4, 2021, 12:17am UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/6 "2021-08-04T00:17:27Z")
Hi @Stephan_Bitterwolf,
Thanks again for your patience! Here are some thoughts on this:
It's hard to tell exactly what the issue is here without knowing the full set up of your experiments or looking at the raw reads/barcodes, but the most likely culprits are:
- The presence of other non-biological nts before your barcodes (which you have already said you noticed). You can add those to the barcode columns so they match exactly.
- Your barcodes may be in some other orientation - try checking whether the complement, or reverse complement of the barcodes match anything in the reads.
- The reads may be in mixed orientation, which _might_ be workable with the ` --p-mixed-orientation` parameter.
Overall though, we wouldn't recommend messing with increasing the error-rate; that starts to enter some shaky territory. q2-demux does support error correction but it is designed for 12nt Golay, not 8 nt Hamming. For 8nt correction you'll have to work with something outside of QIIME 2. QIIME 1 [split\_libraries.py](http://qiime.org/scripts/split_libraries.html) should be able to handle that, which may be a good next step for you to look into.
Hope this helps!
Cheers,
Liz
---
### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [August 4, 2021, 12:17am UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/7 "2021-08-04T00:17:31Z")
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [September 4, 2021, 6:17am UTC](https://forum.qiime2.org/t/barcode-errors-impair-demultiplexing-with-cutadapt/20336/8 "2021-09-04T06:17:40Z")
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