# make.contigs - did not assemble

**URL:** <https://forum.mothur.org/t/make-contigs-did-not-assemble/3084>\
**Category:** Commands in mothur\
**Created:** [January 9, 2017, 5:44pm UTC](https://forum.mothur.org/t/make-contigs-did-not-assemble/3084 "2017-01-09T17:44:34Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![lamab](https://avatars.discourse-cdn.com/v4/letter/l/b5ac83/32.png) [@lamab](https://forum.mothur.org/u/lamab)\
**Post date:** [January 9, 2017, 5:44pm UTC](https://forum.mothur.org/t/make-contigs-did-not-assemble/3084/1 "2017-01-09T17:44:34Z")

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Dear colleagues,

I’m new using Mothur and I’m still learning from tutorials. I am analyzing 50 samples of soil communities, obtained with MiSeq. The data, fastq files, have already been demultiplexed and I’m working with two files per sample foward and reverse (Ex: C2O4\_S33\_L001\_R1\_001.fastq, C2O4\_S33\_L001\_R2\_001.fastq).

I have already followed the following paths:

1. I already set the input and output directories

2. I used the make.file (inputdir = … / raw, type = gz) command to create stability.files

3. I ran make.contigs (file = stability.files, processors = 8). This step occurred satisfactorily.

4. summary.seqs (fasta = stability.trim.contigs.fasta). At that stage is the problem. The sequences were expected to have about 275 bp (v4 region), however, the samples had more than 400 bp (results below). I assumed the reads did not assemble. Any suggestions to solve? I would be grateful if anyone could help.

Start End NBases Ambigs Polymer NumSeqs  
Minimum: 1 298 298 0 3 1  
2.5%-tile: 1 441 441 0 4 167681  
25%-tile: 1 444 444 0 5 1676804  
Median: 1 460 460 0 5 3353608  
75%-tile: 1 467 467 1 6 5030411  
97.5%-tile: 1 471 471 13 8 6539534  
Maximum: 1 602 602 59 301 6707214  
Mean: 1 456.624 456.624 1.50144 5.4271

# of Seqs: 6707214
  
Best Regards,

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**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:** [January 12, 2017, 1:09pm UTC](https://forum.mothur.org/t/make-contigs-did-not-assemble/3084/2 "2017-01-12T13:09:14Z")

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Hi there,

I’d double check with your sequencing center that they really sequenced the V4 region. If you follow the Kozich method your contigs should be ~250 nt. Even if they sequenced off the Illumina adpaters and resequenced the barcodes and primers, the contigs would be shorter than 275 (at this point in the pipeline those should have been removed). If the reads didn’t assemble, you would have 500 nt contigs.

Pat

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<div class="post-metadata">

**Author:** ![lamab](https://avatars.discourse-cdn.com/v4/letter/l/b5ac83/32.png) [@lamab](https://forum.mothur.org/u/lamab)\
**Post date:** [January 12, 2017, 4:32pm UTC](https://forum.mothur.org/t/make-contigs-did-not-assemble/3084/3 "2017-01-12T16:32:41Z")

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Dear Mr. Schloss,

Thank you for your guidance. I checked with the company and they sequenced the v3-v4 region, so the results. I´ll follow the steps about other regions that are in the mothur blog.

Thank you again for your guidance. It was very clarifying and helped me a lot.

Best Regards,