separating fastq files by barcode

Commands in mothur
1

Hello -

When I received a recent miseq 2x250 run back from sequencing, they provided me with two files (Undetermined_S0_L001_R1_001.fastq and Undetermined_S0_L001_R2_001.fastq). I am having trouble separating the two files into individual fastq files for each sample. Any suggestions? The files are of the following format:

@M05104:9:000000000-BFL43:1:1101:15750:1332 1:N:0:CGAGAGTT+CGTCGCTA
TACGGAGGGTGCGAGCGTTAATCGGAATCACTGGGCGTAAAGCGCACGCAGGCGGTCTGTTAAGCTAGATGTGAAAGCCCCGCGCTCAACGTGGGAGGGTCATTTAGAACTGGCAGACTAGAGTCTTGGAGAGGGGAGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGGACATCAATGGCGAAGGCAACTCCCTGGCCAAAGACTGACGCTCATGTGCGGAAGTGTGGGTAGCGAACA
+
BBAABBBBBBBBGFGGGGGGGGHGGGGGHGHHHHHHGGGGHHHGGGGGGGGGGGGGGGHHFGHHGHHHHHHGHEHHHHHHGGCGGGGGHHGGFFGHGGGCGHHHHGGHHHHGHHHGGHHFHHHHHHGHFGHFGGDFAFGHGGGGGGGFGGCFFFGGGGFGGGGGBGGGGGFFFFFFFEFFFF?.9BFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFDFFF.BBFFFD-9@D/BFFFFAFEEFAFFD

Thank you!

2

You will need to create an oligos file, https://mothur.org/wiki/Oligos_File. Do you have the barcode information?

3

Yes, I do have barcode information. I formatted my oligos file as

#primer TATGGTAATTGTGTGYCAGCMGCCGCGGTAA AGTCAGTCAGCCGGACTACNVGGGTWTCTAAT
BARCODE CGAGAGTT ACGACGTG A_0
BARCODE CGAGAGTT ATATACAC FK_13

I then tried running

make.contigs(ffastq=Undetermined_S0_L001_R1_001.fastq, rfastq=Undetermined_S0_L001_R2_001.fastq, oligos=eemb144L.oligos.txt, processors=2)

However, the fasta output from make.contigs was empty.

4

Have you tried running it with bdiffs=1? You could also try setting the checkorient parameter to true? If that doesn’t resolve the issue, could you post some sample sequences so I can troubleshoot it for you?