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Old 11-21-2019, 12:59 AM   #1
Ross_Mcf
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Default MiSeq - issue with index and read 2 (Low Q30)

I recently had a MiSeq run fail and am not 100% sure what caused it.

Here are some details:
Amplicon sequencing run of RNA FFPE samples (125 genes) using a MiSeq V3 (600 cycle) kit loaded at 14 pM. Cluster Density is 909 K/mm2.

Phix spiked in at 15% and sequencing run configured to 131 x 2 paired-end.

SAV Data by Cycle, %Q30: https://imgur.com/a/NZf7Gfb

SAV Run summary: https://imgur.com/a/nQa3sEa

I am leaning towards reagent failure, incorrect run parameters or an issue with the indices.

I would appreciate any help.

Thanks!
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Old 11-21-2019, 04:59 AM   #2
sifekonja
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Seeing as Read 1 was OK (not great, not terrible) and the issues started after that, it might be reagent related or an issue with the system fluidics/optics.

I do not think indexes or run parameters might be a factor here. Just from the Q30 plot the libraries seem to be fine, but %Base from SAV or Bioanalyzer images might help to confirm that.

I think it might be best to show the run to Illumina, a competent support might look at the SAV data (%Base, FWHM) and figure out the problem quickly.
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Old 11-21-2019, 06:09 AM   #3
Ross_Mcf
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Thanks for the answer @sifekonja!

I initially thought the failure might be due to low library or barcode diversity. This is copied from the library prep protocol: "The Illumina sequencers will work best when index diversity within a run is high. For example, if eight samples are included in a run, and the user chooses to use only one MBC Adapter paired with eight different MiSeq Index 1 Primers, the run may fail due to low barcode diversity. In this example it is best to use eight different Archer MBC Adapters paired with eight different MiSeq Index 1 Primers"

My question then would be why is the reverse read so badly affected?

It definitely also looks like it could be a reagent issue.
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Old 11-21-2019, 06:17 AM   #4
Bukowski
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With a 15% PhiX spike in, the last thing it can be described as is 'low diversity'. In fact that's a very high spike in level for more recent MiSeq sequencing protocols for low diversity libraries. Cluster density is low, but in general providing it isn't 'too low' then you should have pretty good quality data. Index diversity I'm not so sure about index diversity, normally you should just follow the guides to add the appropriate diversity in your indexes.. quite spelled out in the Illumina protocols at least.

Contact Illumina support as suggested. If it's a reagent failure, then depending on the nature of the MiSeq support contract, you'll get free reagents to run again.

With regards to why the second read might be affected, remember the order that things are actually sequenced in and what they represent. Read 1, Read 2, Read 3 and Read 4 are in your metrics. Your sequencing reads are Reads 1 and 4 in this context. It does look like the quality tanks on the second index read in particular.

Last edited by Bukowski; 11-21-2019 at 06:24 AM.
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Old 11-21-2019, 06:22 AM   #5
Ross_Mcf
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Hi @Bukowski, thanks for your feedback.

While I fully agree regarding the 15% PhiX and low diversity library I was more concerned with the statement from the protocol about the indices. I will be getting in touch with Illumina.
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Old 11-25-2019, 09:46 AM   #6
itstrieu
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I had a issue with the NextSeq where the Q30 score for read 2 looked horrible and it was determined to be a pump failure.
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