Index R3 fastq file?

[retroant]

Hey Qiime Group.

We are trying to use Qiime to process paired-end 16s data from a MiSeq run and are trying to figure out how the R3/Index fastq file is generated.  We have talked with our sequencing core and they believe that the ability to generate the R3/Index fastq file was retired by Illumina several years ago.  From what I have gathered there might be an option to generate this file through modifying the Reporter.exe.config file.  How do you guys go about generating this third fastq file?   Thanks in advance as it is greatly appreciated! Have a great day.

Doug

[Sarah Owens]

Hi Doug,

We use the MiSeq for 16S amplicons, and use a similar approach for generating fastqs for all of the Earth Microbiome Project samples sequenced at Argonne on the HiSeq. There are a couple ways to generate this fastq. One way is to use an older version of MiSeq reporter than you're currently using, we haven't upgraded our MSR (we're using version 1.1.6) so that we can continue to generate the index reads automatically. Newer versions don't give you the three files. If everything is updated on your MiSeq, you could requeue the run using the older version of MiSeq reporter on a PC to generate these index reads. 
Another way is to use CASAVA 1.8.2 to produce your reads. To do this, you use the function --use-bases-mask Y*, Y*, Y*  This function will generate 3 reads (it treats your index read as just another read). Treating the read as an index read confuses CASAVA (tries to demultiplex, etc.), so treating it as 3 reads is the best way to go.

Sarah

Sarah M. Owens
Technical Director, IGSB-NGS Core
Argonne National Laboratory
9700 S. Cass Avenue
Bldg. 202, Rm. A353
Lemont, IL 60439
Sarah...@anl.gov
630.252.2101

[retroant]

Hi Sarah.

Thank you very much for your response.  I will run this information by our sequencing core and see what we might be able to do. I would assume (possibly incorrectly) that we will need to do another run in order to implement these changes?  Is it possible to generate the index fastq file from data that has already been sequenced?  Thanks again!

Doug

[Owens, Sarah]

Hi Doug,

Neither of these changes requires you to re-run the sequencing run. You can requeue the fastq file generation in MiSeq Reporter on a PC that your sequencing core probably already has set-up for their MiSeq. You'd use the current run folder to generate fastqs using CASAVA 1.8.2 as well.

Thanks.
Sarah

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Sarah M. Owens, M.S.