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Old 11-06-2012, 12:31 PM   #1
scotoma
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Location: Seattle

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Default Miseq index reads missing

Hello, I was just wondering if anyone has had this problem before. I recently tried to sequence a library containing 4 barcodes on the Miseq (using custom forward, reverse, and index primers and the 2x151 kit). On my sample sheet (below), I only put 8 Ns (the length of my barcode) under "Index" since I was just going to manually demultiplex later. However, it looks like only 302 cycles completed since there are only 302 folders in the BaseCalls/L001 folder. Does this mean that the index run never completed? If not, can I still recover those reads?

Using Illumina RTA 1.13.56

[Header]
Investigator Name D
Project Name t
Experiment Name t2
Date 11/1/12
Workflow Resequencing
Assay TruSeq DNA/RNA
Description
Chemistry Default
[Reads]
151
151
[Settings]
CustomRead1PrimerMix C1
CustomIndexPrimerMix C2
CustomRead2PrimerMix C3
OnlyGenerateFASTQ 1
[Data]
Sample_ID Sample_Name Sample_Plate Sample_Well Sample_Project Index Description GenomeFolder
t2 t2 NA NA t NNNNNNNN first_try_at_t C:\Illumina\MiSeq Reporter\Genomes\PhiX\Illumina\RTA\Sequence\Chromosomes

I couldn't seem to find the answer to this question elsewhere, but sorry if I missed it and thank you for your help.
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Old 11-06-2012, 01:33 PM   #2
hoisinjl
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Default

Can you look at the RunInfo.xml file (in the runfolder). If the software correctly parsed your samplesheet, it should read:

<Reads>
<Read NumCycles="151" Number="1" IsIndexedRead="N" />
<Read NumCycles="7" Number="2" IsIndexedRead="Y" />
<Read NumCycles="151" Number="3" IsIndexedRead="N" />
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Old 11-06-2012, 01:48 PM   #3
Number6
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The MiSeq treats barcode reads differently than the HiSeq and one way to do this is to modify the MiSeq Reporter.exe.config file to include the "CreateFastqForIndexReads" setting. This is described in the MiSeq Reporter User Guide in the "MiSeq configurable settings" section.

This will give you the barcode reads as a separate fastq file, instead of being in the sequence header, like the HiSeq.

You can modify this file, restart the MiSeq Reporter service, and reanalyze your run with MSR.
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Old 11-06-2012, 02:02 PM   #4
scotoma
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Thank you both for your help. Unfortunately, RunInfo.xml looks like this:

<Reads>
<Read NumCycles="151" Number="1" IsIndexedRead="N" />
<Read NumCycles="151" Number="2" IsIndexedRead="N" />
</Reads>
<FlowcellLayout LaneCount="1" SurfaceCount="1" SwathCount="1" TileCount="12" />


so it appears MSR parsed the sample sheet incorrectly and the index read was skipped. Regardless, I'll try Number6's strategy of configuring Miseq Reporter to output a separate fastq for index reads (if not for this run, at least for the next). Also, I guess that in the future I'll input the actual index sequences. Is there anything else I should try? Thanks again.
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Old 11-06-2012, 02:12 PM   #5
hoisinjl
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I am also going to try the "CreateFastqForIndexReads" as that will solve some of the post-analysis work I do to generate the three files.

When starting a run, after you upload the SampleSheet, the next screen shows how the SampleSheet is parsed. I would add that you should double-check that the # cycles for read1, index and read2 are correctly read.
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