# Demultiplexing MiSeq Data **URL:** \ **Category:** Commands in mothur\ **Created:** [September 3, 2014, 6:01pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004 "2014-09-03T18:01:19Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![ericstaunton](https://avatars.discourse-cdn.com/v4/letter/e/ecc23a/32.png) [@ericstaunton](https://forum.mothur.org/u/ericstaunton)\ **Post date:** [September 3, 2014, 6:01pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004/1 "2014-09-03T18:01:19Z")
Hello, I’m trying to separate data from a barcoded MiSeq run into separate groups. The sequencing service I’m using used barcodes on the forward primer only, and I made an oligos file with the barcodes and primers (below). When I run the make.contigs command (using fasta=stability.files and the .files file has the three columns as described in the MiSeq SOP wiki) I get an error that says “barcodes must be paired unless you are using an index file.” I’ve searched though all the files I have, but I can’t find anything that looks like the example index file on the make.contigs wiki page. Is this something I’m meant to make? If so, can someone elaborate on how? Thanks! Let me know if you need more information! Eric primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT V3 barcode GAGAGTGT None HogWaste1 barcode GAGTCACT None HogWaste2 barcode GAGATCAG None HRSD1 barcode GAGATCTC None HRSD2 barcode GAGATGAC None Red1 barcode GAGATGTG None Red2 barcode GAGTACAG None Brown1 barcode GAGTACTC None Brown2 barcode GAGTAGAC None Floc1 barcode GAGTCAGA None Floc2 barcode GAGTAGTG None Eff1 barcode GAGTCTCA None Eff2 ---
**Author:** ![ericstaunton](https://avatars.discourse-cdn.com/v4/letter/e/ecc23a/32.png) [@ericstaunton](https://forum.mothur.org/u/ericstaunton)\ **Post date:** [September 3, 2014, 7:47pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004/2 "2014-09-03T19:47:36Z")
I’ve continued going through files and found something that looks very similar to the example index file, but it’s not fastq, the sequences are much longer than in the example, and the header isn’t in the same format (first few lines pasted below). The first 8 bases do correspond to my barcodes, but I’m not sure if this is my index file or if it somehow becomes my index file. Thank you! > M02542:21:000000000-AAMB0:1:2105:21214:20751 1:N:0:7 > GAGTAGACGTGCCAGCCGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGATTTACTGGGCGTAAAGGGTGCGTAGGTGGTTTCTTAAGTCAGAGGTGAAAGGCTACGGCTCAACCGTAGTAAGCCTTTGAAACTGGGAAACTTGAGTGCAGGAGAGGAGAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTTGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCGTGTAGTCC > M02542:21:000000000-AAMB0:1:2116:9375:7377 1:N:0:7 > GAGTAGTGGTGCCAGCCGCCGCGGTAATACGTAGGGGGCGAGCGTTGTCCGGATTTACTGGGTGTAAAGGGCGCGTAGGCGGAACGGTAAGTCAGTGGTGAAATCTTGCGGCTTAACCGCAAAACTGCCGTTGATACTGCTGTTCTTGAGTATAGGAGAGAGAGGCGGAATTCCAGGTGTAGTGGTGAAATACGTAGATATCTGGAAGAACACCAATTGCGAAGGCTGCTTCTTGGCCTATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCCGTAGTCC ---
**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:** [September 10, 2014, 5:54pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004/3 "2014-09-10T17:54:23Z")
Can you check this thread and see if it solves your problem? [http://www.mothur.org/forum/viewtopic.php?f=3&t=3159&start=10#p9096](http://www.mothur.org/forum/viewtopic.php?f=3&t=3159&start=10#p9096) Pat ---
**Author:** ![Rich](https://avatars.discourse-cdn.com/v4/letter/r/35a633/32.png) [@Rich](https://forum.mothur.org/u/Rich)\ **Post date:** [February 3, 2015, 5:03pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004/4 "2015-02-03T17:03:56Z")
Dear Mothur, I am getting more of scrap sequences than contigs. I guess the problem could be oligos file. Please find it below mothur “#make.contigs(ffastq=F\_001.fastq, rfastq=R\_001.fastq,oligos=exp1.oligos, processors=1)” primer ACTCCTACGGGAGGCAGCAG GGACTACHVGGGTWTCTAAT barcode ACAGCCACCCATCGA CCATCACATAGG Saga2 barcode ACAGCCACCCATCGA GTGGTATGGGAGA Saga3 barcode ACAGCCACCCATCGA ACTTTAAGGGTGA Saga4 barcode ACAGCCACCCATCGA TGTTGCGTTTCTTC Saga5 barcode ACAGCCACCCATCGA ATGTCCGACCAATC Saga6 If my oligo file format is correct then what can be the possible reason of th problem? Best, Rich ---
**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:** [February 4, 2015, 3:05pm UTC](https://forum.mothur.org/t/demultiplexing-miseq-data/2004/5 "2015-02-04T15:05:51Z")
Have you tried running it with pdiffs=2, bdiffs=1?