# Oligos file in make.contigs

**URL:** <https://forum.mothur.org/t/oligos-file-in-make-contigs/2948>\
**Category:** Commands in mothur\
**Created:** [August 11, 2016, 8:47am UTC](https://forum.mothur.org/t/oligos-file-in-make-contigs/2948 "2016-08-11T08:47:42Z")\
**Posts on this page:** 2\
**Page:** 1

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**Author:** ![evensr](https://avatars.discourse-cdn.com/v4/letter/e/c67d28/32.png) [@evensr](https://forum.mothur.org/u/evensr)\
**Post date:** [August 11, 2016, 8:47am UTC](https://forum.mothur.org/t/oligos-file-in-make-contigs/2948/1 "2016-08-11T08:47:42Z")

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Dear all,

I have a question regarding the use of an oligos file in the make.contigs step. So far in my amplicon analysis, I have not removed the primer sequences (including the barcode in the reverse primer) in the beginning of the pipeline, due to the assumption that these will be trimmed away during alignment against the Silva 16S database.

I suspect I have quite many «raw» reads with \>1 mismatch to the barcode sequence, and now I want those to not be assembled during «make.contigs». As I understand, it’s possible to provide the primer and barcode sequences in an «oligos file» in this command, and specify the amount of allowed mismatches (I want 0 to the barcodes) to these sequences. I use the Caporaso primers (used by Earth Microbiome Project), which have a 12 bp Golay barcode in the reverse primer, and also spacer and linker (see ftp://ftp.metagenomics.anl.gov/data/misc/EMP/SupplementaryFile1\_barcoded\_primers\_515F\_806R.txt). My oligos file is therefore like shown below (tab separated). When used, I get no assembled contigs at all. When not used, I get a lot of contigs assembled.

Why isn’t my oligos file working in the “make.contigs” step?

primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT V4  
barcode none GGAGACAAGGGA L0814\_25G1  
barcode none AATCAGTCTCGT L0814\_25G2  
barcode none AATCCGTACAGC L0814\_25G3  
barcode none CCAGTGTATGCA L0814\_27G1  
barcode none CCTCGTTCGACT L0814\_27G2  
barcode none TGAGTCACTGGT L0814\_27G3  
barcode none CACGCCATAATG L0814\_28G1  
barcode none CAGGCGTATTGG L0814\_28G2  
barcode none GGATCGCAGATC L0814\_28G3

Kind regards,

Even

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**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:** [August 11, 2016, 4:28pm UTC](https://forum.mothur.org/t/oligos-file-in-make-contigs/2948/2 "2016-08-11T16:28:17Z")

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Could you post some forward and reverse reads so I can help you figure out what is causing the issue?