Make.contigs help

Commands in mothur
1

I am very new so I don’t know if this is an issue or just me being clueless.

I am trying to run make.contigs but just get lines and lines of Warning messages and no group/count output. I have the pairs fastq.gz files that I have trimmed using cutadapt in the mothur folder. So for example I have my pairs as “trimmed_BP1_1.fastq.gz” and “trimmed_BP1_2.fastq.gz”. I then used make.file to make the stability.files (which seems to have worked).

I then run make.contigs(file=stability.files, processors=8) but just get lines and lines of this (example except):

[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_13609_28642.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_13676_28685.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_15309_28704.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_18097_28723.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_18186_28772.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_18909_28809.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_16906_28850.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_17719_28857.
[WARNING]: name mismatch in forward and reverse fastq file. Ignoring, M05898_65_000000000-CC66F_1_1114_15438_28866

What am I doing wrong? Do the files need to live somewhere else?

I am running mothur v.1.44.3

I also posted this in on github/mothur

2

likely cutadapt removed whole sequences in the R1 but not in the R2, and viceversa, so the names of the reads do not agree between the two files.

Do you need to run cutadapt before? You can do that in mothur

3

What @leocadio said. There’s really no reason to use cutadapt. We find that quality trimming the reads before make.contigs makes the quality of the assembly worse, not better.

4

Thank you both - I am very new to this and am working off protocol written by a colleague who has used Mothur before. Her protocol uses cutadapt. So, just to clarify, I run make.contigs first and then trim.seqs?

Edit: typo.

5

follow up: i ran make.contigs (with no issues) and then trim.seqs using the stability.trim.contigs.fasta file. I made a .oligos file containing the forward and reverse, but the output is empty - the files have nothing in them. What am I doing wrong?

6

I also tried make.contigs(file=stability.files, oligos=primers.oligos, processors=1)

My primers.oligos file contains:

forward CCTACGGGAGGCAGCAG
reverse ATTACCGCGGCTGCTGG

The output from that is also empty.

7

ran it again. all okay. having an issue with unique.seqs but will open another thread to keep things clear.

1 Like
8

Sorry I was not receiving the alerts from new messages… Have you solved the make.contigs, then?

9

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