# Sequence Length **URL:** \ **Category:** Theory behind mothur\ **Created:** [May 2, 2017, 9:03pm UTC](https://forum.mothur.org/t/sequence-length/3236 "2017-05-02T21:03:46Z")\ **Posts on this page:** 1\ **Showing post:** 10
**Author:** ![jlawrence](https://avatars.discourse-cdn.com/v4/letter/j/838e76/32.png) [@jlawrence](https://forum.mothur.org/u/jlawrence)\ **Post date:** [May 30, 2019, 1:57am UTC](https://forum.mothur.org/t/sequence-length/3236/10 "2019-05-30T01:57:05Z")
Hello, I have some followup questions on this topic. I similarly have V4 .fastq data from a 2x250 MiSeq run and it seems I too need to remove the primers (my reads were also assembling to ~290). I have 28 .fastq files for 14 samples and am following the MiSeq SOP. My oligos.txt file looks like this currently: > primer GTGYCAGCMGCCGCGGTAA GGACTACNVGGGTWTCTAAT > # BARCODE none none JG01 > # BARCODE none none JG02 > …etc. > make.contigs(file=stability.files, oligos=oligos.txt, processors=4) Now reads assemble to the expected 253 bp length instead of 292, but I still have a few questions: 1. Do I need to account for degeneracy (if that’s the correct term) in these primers by listing all the possible permutations in my oligos file? ([e.g this post](https://forum.mothur.org/t/removing-sequence-primers/2269)) 2. make.contigs() throws a warning: \* **[WARNING]: your oligos file does not contain any group names. mothur will not create a groupfile.** \*. How do I work around this if barcodes are already removed and each sample is split into forward and reverse fastq files? Without a groupfile, do all the sequences end up in the same group, even if stability.files has the sample IDs? 3. Could leaving the primers in previously be a cause for why my batch script was crashing during cluster.split() - perhaps the primers caused excessive unique sequences ([per the blog post here](http://blog.mothur.org/2014/09/11/Why-such-a-large-distance-matrix/))? Appreciate any suggestions. --- _[View the full topic](https://forum.mothur.org/t/sequence-length/3236)._