make.contigs multiple warnings

Commands in mothur
1

We have previously followed the Mothur SOP for wet lab and MiSeq SOP for processing / analysis. This worked very last last time. However, in a recent run of 192 samples the make.contigs commands is throwing back numerous warnings. Once the make.contigs command is initiated it runs seemingly fine for about 10 minutes then the following appears…

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[WARNING]: reading CCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACG expected a name with + as a leading character, ignoring.[WARNING]: names do not match. read M01

2

FHHHHHHHHHHHGHHGGEEGHHBHHGGGFGDH########?#################<??FFGHHFHHHHGHGHH##.###>>############.<;:.:####;=:###9;.#.;;A9#########;;;:;BFD;@EEEFFAAAFF.;:9BFFF9-9BFAF-@DE.ABBFF9BF. for fasta and CCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACG for quality, ignoring.[WARNING]: Lengths do not match for sequence M01

3

For some reason I cannot paste the whole list of issues. I have log files and will be happy to attach (if possible) or email. Let me know.

Thanks advance,
Chris

4

Looks like an error in the fastq file. Can you use grep to see if there is indeed some sort issue with the input file?

5

Thanks for the response. I will do this ASAP. To confirm, to use grep on a MAC I will open the terminal and cd the folder with the fastq files and run ‘grep -c “>” *.fastq’?

Thanks,
Chris

6

grep -C 50 “STRINGTOMATCH” filename You probably want a longer string than > to avoid alot of grep matches.

7

Thank you! We identified 10 fastq files which were causing the issue. Removing these files from stability.files allowing the make.contigs command to run with no errors.

Any ideas on what was causing the issue? There is nothing striking about files that were corrupt that jumps out and this has never happened before…

Thanks,
Chris

8

It may not be the entire file. Perhaps just part of the file just corrupted? Hard to say why. You could ask for another copy from your provider.