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Hello,

I recently received data back to analyze wolf diet composition from scat samples. The data contained 384 dual-indexed samples. Using OBITools3 (Version 3.0.1b18), I have been able to import my fastq files, alignpairedend, and filter based on alignment score, but I am having difficulty with demultiplexing the data. When I run the following command:

obi ngsfilter -t wolf/ngsfile -u wolf/unidentified_sequences wolf/good_sequences wolf/identified_sequences

It runs, but the output is zero remaining sequences. I am still unsure if I am formatting my ngsfile correctly and have included the first line of the file below:

TX1 MTU_001_1 CGAGAGTT:ATCGTACG TTAGATACCCCACTATGC TAGAACAGGCTCCTCTAG F @

In the third column of the ngsfile, should the i5 or the i7 index be listed first, and do either of the indexes need to be reported in the reverse complement?

I also used Illumina's BaseSpace software to export my data already demultiplexed to make sure that my data contained indexes. I was able to retrieve most of the samples where I received a fastq file for each sample. I tried running the OBITools3 pipeline on a single fastq file from a single sample, but ran into the same issue as before where the ngsfilter command resulted in zero remaining reads. I used the following for the ngsfile:

TX1 MTU_001_1 -:- TTAGATACCCCACTATGC TAGAACAGGCTCCTCTAG F @

Thanks in advance,

Sam