Running make.file mothur crashes

[luizper]

Hey guys, I’m trying to run mothur for the first time on my dataset (I’m trying to follow the MiSeq SOP tutorial but using my sequencing data). But during the first step, the make.file(inputdir=., type=fastq, prefix=stability), I put my fastq files on the folder and run the command but mothur crashes. When I try to run using the MiSeq SOP fastq data it works just fine, so I was looking at my fastq files and it seems that it has a different format.

Here is a short example from my R1 read:

@M02526:311:000000000-J348V:1:1101:12313:2025 1:N:0:CAGATTC+CCATGGC
TTCGTCCTACGGGGGGCAGCAGTGGGGAATCTTGGGCAATGGGCGAAAGCCTGACCCAGCGACACTGCGTGGGTGATGAAGATCTTTGGATTGTAAAACCCTTTTCCTGAAGATGAAAGGTAGGATTGAGAATATCCTTTCTACCCTGACAGTATTCAGGGAATAAGCCCCAGCCAACTACGTGCCAGCAGCTGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTACTGGGTGTGAAGGGTGTGCAGGCTTCCTTTTAAGTTGATTGTGAAATCCAAGAGGCTCAACCTCTGAATTTCAGT
+
CBCCCEGGGG7@FC7:FCGD@<F<8E7CEFEFG99E87FDCDF:6=>AFGF8EGFFFDF7>FFFFFCF:@@CECF<9A,<>FGFGCC8FGDG9FAFBBEGGGGCFCC9@@9FGFFFG>GFFG9FGGC9BCFGGGGGGGGGFGGGGDDGFGECFGFCEG75CF9C9C8BEEDGGGGGGGGEGDGFGGGFCFGGGCDGGGGGGGFGG*9>33CF3CDGF<>CDDGCDFFFCFFFDFFDBFA<F0?1;>FAF4>FFBFFFFF>7>AD2<>AF>442:@BB>>4116::B<4:?7?B?A2AA)644.
@M02526:311:000000000-J348V:1:1101:18486:2043 1:N:0:CAGATTC+CCATGGC
TCCTACGGGACGCAGCAGTGAGGAATATTGGTCAATGGGCGCAATCCTGAGCCAGCCATCCCGCGTGCAGGAAGACGGCGCTATGCGTTGTAAACTGCTTTTCCAGGGGGAGAAATTTCCGTACGTGTACGGAATTGACGGTACCCTGGGAATAAGCATCGACTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAAGCGTTATCCGGATTCATTGGGTTTAAAGGGTGCGTAGGCGGACTATTAAGTCAGTGGTGAAATCCTGCAGCTTAACTGCAGAACTGCCATTGATACTGCTGC
+

Whereas an example from the F3D0_S188_L001_R1_001.fastq obtained from the MiseqSOP dataset:
@M00967:43:000000000-A3JHG:1:1101:18327:1699 1:N:0:188
NACGGAGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTGCCAAGTCAGCGGTAAAATTGCGGGGCTCAACCCCGTACAGCCGTTGAAACTGCCGGGCTCGAGTGGGCGAGAAGTATGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGATATCACGCAGAACCCCGATTGCGAAGGCAGCATACCGGCGCCCTACTGACGCTGAGGCACGAAAGTGCGGGGATCAAACAG
+
#>>AABABBFFFGGGGGGGGGGGGGGGGHHHHHHHGGGHHHHHGHGGGGGGGHGGGGGGHHHHHHHHHHGGGGGHHHHGHGGGGGGHHBGHGDGGGGGHHHGGGGHHHHHHHHGGGGGHG@DHHGHEGGGGGGBFGGEGGGGGGGG.DFEFFFFFFFDCFFFFFFFFFFFFFFFFFFFFFFFFFFDFDFFFEFFCFF?FDFFFFFFFFAFFFFFFFFFFFBDDFFFFFEFADFFFFFBAFFFA?EFFFBFF
@M00967:43:000000000-A3JHG:1:1101:14069:1827 1:N:0:188
TACGGAGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTGCCAAGTCAGCGGTAAAATTGCGGGGCTCAACCCCGTACAGCCGTTGAAACTGCCGGGCTCGAGTGGGCGAGAAGTATGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGATATCACGCAGAACCCCGATTGCGAAGGCAGCATACCGGCGCCCTACTGACGCTGAGGCACGAAAGTGCGGGGATCAAACAG
+

Do you guys know how can I solve this? Many thanks!

[westcott]

The make.file command does not read the fastq files so the formatting should not matter. Make.file looks for fastq files and creates and file file containing the paired fastq files.

Some troubleshooting ideas:

Are you using our current version 1.48.1?
(Run “./mothur --version” to find your version)

Do you have read permissions for the fastq files?
(Run “ls -l” to check permissions on the input folder)

Is there additional information being outputted in the log file?