# oligos in make.contigs

**URL:** <https://forum.mothur.org/t/oligos-in-make-contigs/1886>\
**Category:** Commands in mothur\
**Created:** [June 11, 2014, 8:21am UTC](https://forum.mothur.org/t/oligos-in-make-contigs/1886 "2014-06-11T08:21:07Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![awil](https://avatars.discourse-cdn.com/v4/letter/a/f05b48/32.png) [@awil](https://forum.mothur.org/u/awil)\
**Post date:** [June 11, 2014, 8:21am UTC](https://forum.mothur.org/t/oligos-in-make-contigs/1886/1 "2014-06-11T08:21:07Z")

</div>

Hello,

I got reads from MiSeq and then I am following for the analysis in MiSeq SOP protocol. I understand that fastq in this MiSeq SOP protocol were seperated into each source. However, I have some problem to split barcodes for each source because all sources is pooled within one experiment (like as 454, which I normally use trim.seqs with oligo file in function allfiles=T)

This is the sample in my oligos file :

> primer AGAAACGTGAGCGCTG ATACTAGGGTATTTAA  
> BARCODE ACTATGTG NONE water1  
> BARCODE TGTACACG NONE water2  
> BARCODE TACTATGG NONE sand1  
> BARCODE ATAGCACT NONE sand2  
> BARCODE AATCGTAG NONE water3  
> …

I used command to make contigs and separate oligos in below :

```plaintext
make.contigs(ffastq=POOL_S1_L001_R1_001.fastq, rfastq=POOL_S1_L001_R2_001.fastq, oligos=file.oligos, processors=8)

```

I got these errors :

> > > > > > Processing POOL\_S1\_L001\_R1\_001.0ffastatemp (file 1 of 1) \<\<\<\<\<  
> > > > > > [ERROR]: barcodes must be paired unless you are using an index file.  
> > > > > > [ERROR]: barcodes must be paired unless you are using an index file.  
> > > > > > [ERROR]: barcodes must be paired unless you are using an index file.  
> > > > > > …

Could you please suggest me?  
Thank you very much.  
Alisa

---

<div class="post-metadata">

**Author:** ![awil](https://avatars.discourse-cdn.com/v4/letter/a/f05b48/32.png) [@awil](https://forum.mothur.org/u/awil)\
**Post date:** [June 11, 2014, 11:53am UTC](https://forum.mothur.org/t/oligos-in-make-contigs/1886/2 "2014-06-11T11:53:48Z")

</div>

Hello,

I tried to use this command :

```nohighlight
make.contigs(ffastq=POOL_S1_L001_R1_001.fastq, rfastq=POOL_S1_L001_R2_001.fastq)

```

After that I got trim.contig.fasta which they combined R1 and R2 together, their barcodes and primers are still appeared in new contigs.  
However, I think that I should use trim.seqs from 454 SOP protocol again to trim their barcodes and primers off and split in each source. How do you think about this?

Thank you very much.