# Cannot demultiplex fastq files with barcodes in the header **URL:** \ **Category:** Commands in mothur\ **Created:** [November 3, 2020, 4:17pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748 "2020-11-03T16:17:46Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![Sci\_evol](https://avatars.discourse-cdn.com/v4/letter/s/e95f7d/32.png) [@Sci\_evol](https://forum.mothur.org/u/Sci_evol)\ **Post date:** [November 3, 2020, 4:17pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748/1 "2020-11-03T16:17:46Z")
Dear mothur team, I am trying to demultiplex the seq files of a MiSeq run, which have forward.fastq, reverse.fastq and one index.fastq. **example lines from the forward.fastq:** @MISEQ:192:000000000-A88BF:1:1101:14475:1417 1:N:0:CCCTCTTTTTTC TCCTTCTTCTCCTCTCTTTCTCCTTCTTTCTTTTTTTTCCCTTTCTCTTCTTCTTTTTTTTCCTTCCTTTTTTCCCTTTTTCTTCTCCCCCTTTCCCTTTCCTTTTCTCCTTTTTTTCTTTCTTTCCTTTTCTTTCTTCTTCCTTCCTTTTTTCTCTTTTCCCTTCTTCTCTCTCCCTCCTTCCTCCTCTTTCTCCTTCCTCTTCCTTTCCTTCCCCTTCCTCTTCTTCTCTCCCTCTTTTTTCTCCCCCC **example lines from the reverse.fastq:** @MISEQ:192:000000000-A88BF:1:1101:16318:1652 1:N:0:CACGCCATAATG TCCTTCTTCTCCTCTCTTTCTCCTTCTTTCTTTTTTTTCCCTTTCTCTTCTTCTTTTTTTTCCTTCTTTTTTTCCCTTTTTCTTCTCCCCCTTTTCCCTTCTTTTCGTCCTTGTTTCCTTTCTTTCGCTCTATTTCTTCTTCCTTCCTTTTTTCTCTTTTCCCTTCTTCGCTCTCCCGCCGCACTCCACTTTCTCCTGCCTCTTCCTCCCCTTCCCCTTCCTCTTCTTCTCTCCCTTTTTTTTCTCCCCCC **example lines from the index.fastq:** @MISEQ:192:000000000-A88BF:1:1101:14475:1417 1:N:0:CCCTCTTTTTTC CCCTCTTTTTTC + AAAAAAAAAAAA @MISEQ:192:000000000-A88BF:1:1101:15818:1635 1:N:0:CTCCCCTCCTCT CTCCCCTCCTCT However, I couldn’t figure out how I should demultiplex this kind of data. Would you please help me with this? Thank you very much for your help. ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [November 3, 2020, 5:46pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748/2 "2020-11-03T17:46:47Z")
Hi there - In [`make.contigs`](https://mothur.org/wiki/make.contigs/#findex--rindex) you will use `ffastq`, `rfastq`, and `findex` (or `rindex`). You’ll also need to provide an oligos file to tell `make.contigs` which index goes with each sample. Take a look at the wiki page, see if you can figure it out. If not, come on back and let us know how far you got so we can help with the next steps. Thanks, Pat ---
**Author:** ![Sci\_evol](https://avatars.discourse-cdn.com/v4/letter/s/e95f7d/32.png) [@Sci\_evol](https://forum.mothur.org/u/Sci_evol)\ **Post date:** [November 11, 2020, 9:11pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748/3 "2020-11-11T21:11:20Z")
Dear Pat, Thank you very much for your reply. I am trying since then. However I am having an error with the **following command** : make.contigs(ffastq=fwd.1.fq, rfastq=rev.2.fq,findex=index.mid.fq, rindex=index.mid.fq, oligos=oligos.txt, bdiffs=2, pdiffs=2,processors=12) **My oligos file looks like:** forward ACACGTCTGAACTCCAGTCAC f\_pr reverse GTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT r\_pr barcode TATCGTTGACCA TATCGTTGACCA Sample1 barcode CCAGTGTATGCA CCAGTGTATGCA Sample2 barcode TGAGTCACTGGT TGAGTCACTGGT Sample3 barcode ACACCTGGTGAT ACACCTGGTGAT Sample4 **A part from the error message:** [WARNING]: R\_PR is not recognized as a valid type. Choices are forward, reverse, and barcode. Ignoring BARCODE. [WARNING]: TATCGTTGACCA is not recognized as a valid type. Choices are forward, reverse, and barcode. Ignoring TATCGTTGACCA. [WARNING]: Sample1 is not recognized as a valid type. Choices are forward, reverse, and barcode. Ignoring BARCODE. Any idea? Many thanks. ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [November 16, 2020, 1:15pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748/4 "2020-11-16T13:15:02Z")
Mothur does not allow reverse primers to have names. [https://mothur.org/wiki/oligos\_file/](https://mothur.org/wiki/oligos_file/) Your reverse primer has a name of r\_pr, which is causing the read of the oligos file to fail. Try this instead: **My oligos file looks like:** primer ACACGTCTGAACTCCAGTCAC GTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT desiredPrimerName barcode TATCGTTGACCA TATCGTTGACCA Sample1 barcode CCAGTGTATGCA CCAGTGTATGCA Sample2 barcode TGAGTCACTGGT TGAGTCACTGGT Sample3 barcode ACACCTGGTGAT ACACCTGGTGAT Sample4 Also, I noticed the forward and reverse index files have the same name. The forward index file should contain the forward barcodes, and the reverse index file should contain the reverse barcodes. make.contigs(ffastq=fwd.1.fq, rfastq=rev.2.fq,findex= **index.mid.fq** , rindex= **index.mid.fq** , oligos=oligos.txt, bdiffs=2, pdiffs=2,processors=12) ---
**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\ **Post date:** [November 26, 2020, 1:15pm UTC](https://forum.mothur.org/t/cannot-demultiplex-fastq-files-with-barcodes-in-the-header/20748/5 "2020-11-26T13:15:08Z")
This topic was automatically closed 10 days after the last reply. New replies are no longer allowed.