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Old 04-18-2019, 12:02 AM   #1
JuliaRed
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Location: Austria

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Default MiSeq-no reads mapped to index ID

Hello there,

recently I performed a MiSeq run (v3 kit, standard flow cell) with 40 samples and faced some difficulties. First of all, we forgot to add the sample sheet before the run. We added it after the run had finished and data were analysed just fine.
But now I am looking at my data and got quite bad results. Overall the cluster density was low (390 K/mm2), we usually have cluster densities of ~800-1000 K/mm2. But the most puzzling problem is that the MiSeq seems to have difficulties with the indexing. In the Sequence Analysis Viewer in the tab "Indexing" no indices are present, so the MiSeq tells me that no reads were mapped to Index Id. However, when I perform sequence analysis, some samples can be analysed, even though most of the samples can not. The Illumina Tech Support told me, that some of my samples were not demultiplexed.
Do you have any idea what went wrong or if/how I can fix this problem? My hope is that the data is there but only has to be re-analysed somehow.

Best regards
Julia
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Old 04-18-2019, 04:21 AM   #2
GenoMax
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On your MiSeq go to "D:\Illumina\MiSeq Analysis\Flowcell_ID\Unaligned\Reports\html" directory. There should be an index.html file there. Open it using a browser on MiSeq. Scroll all the way to the bottom of the page. It should show top 10 indexes the sequencer sequenced. Compare them against what you thought should have been the indexes. It may just be a matter of reconciling the indexes in your samplesheet and re-running MiSeq reporter to retrieve your data.

That said, if there are N's etc in the indexes then it may be an issue. Considering cluster density that should not be a problem.

Did tech support not work with you on this?
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Old 04-18-2019, 07:06 AM   #3
kmcarr
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Quote:
Originally Posted by JuliaRed View Post
...First of all, we forgot to add the sample sheet before the run. We added it after the run had finished and data were analysed just fine...
Julia,

Pardon what may be a stupid question but how did you get the run started without a sample sheet? The MiSeq requires a sample sheet matching the reagent cartridge name and if it can't find one setup won't proceed. Also, since it is the sample sheet which tells the MiSeq the format and length of the index reads required was a proper index read performed?
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Old 04-19-2019, 12:28 AM   #4
JuliaRed
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Quote:
Originally Posted by kmcarr View Post
Julia,

Pardon what may be a stupid question but how did you get the run started without a sample sheet? The MiSeq requires a sample sheet matching the reagent cartridge name and if it can't find one setup won't proceed. Also, since it is the sample sheet which tells the MiSeq the format and length of the index reads required was a proper index read performed?
This is true, I did not describe the problem properly. We had the sample sheet on a USB stick, but the stick was removed immediately after the run had started. So the MiSeq had the information on how to perform the run.
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