MiSeq photobleaching

[esherman]

I've noticed that even when accounting for kit expiration date, cluster density, %PF, phasing, and prephasing, some of our runs have excellent Q-scores all the way until the end of the read (occasionally 95%+ of all bases are >Q30) and some runs just barely pass spec (75% of all bases >Q30). I'm specifically looking at V2 chemistry 2x250 kits.

I've also noticed that the signal/noise ratio seems to be extremely highly predictive of Q-scores, so I'm wondering if the less than stellar runs were the result of photobleaching of the incorporation mix (presumably the one containing fluorescently tagged nucleotides given the purple color)

During training, we were told to thaw the reagent cartridge in cold water, but nothing was mentioned regarding a light or dark environment, so we've just been keeping the cartridge in a water bath that was not protected from light. We typically thaw our cartridges for 'about' an hour, so there's no strict standard that's followed. Now I'm finding literature suggesting that NextSeq cartridges are thawed in the dark. Does anyone have any experience regarding Q-scores obtained when thawing MiSeq cartridges in the dark or would this be an unnecessary precaution?

[pmiguel]

I've noticed that even when accounting for kit expiration date, cluster density, %PF, phasing, and prephasing, some of our runs have excellent Q-scores all the way until the end of the read (occasionally 95%+ of all bases are >Q30) and some runs just barely pass spec (75% of all bases >Q30). I'm specifically looking at V2 chemistry 2x250 kits.

I've also noticed that the signal/noise ratio seems to be extremely highly predictive of Q-scores,

This is probably because Q-scores calculationsuse signal/noise as parameters.

so I'm wondering if the less than stellar runs were the result of photobleaching of the incorporation mix (presumably the one containing fluorescently tagged nucleotides given the purple color)

I'm not sure what the purple color comes from, but the dyes-labels for the terminators is probably the same as those used for the HiSeq, where the mix is a pink color.
If Illumina thought photo bleaching was an issue I'm guessing they would have recommended thawing the reagents in the dark.

During training, we were told to thaw the reagent cartridge in cold water, but nothing was mentioned regarding a light or dark environment, so we've just been keeping the cartridge in a water bath that was not protected from light. We typically thaw our cartridges for 'about' an hour, so there's no strict standard that's followed. Now I'm finding literature suggesting that NextSeq cartridges are thawed in the dark. Does anyone have any experience regarding Q-scores obtained when thawing MiSeq cartridges in the dark or would this be an unnecessary precaution?

The NextSeq uses different fluors, so Illumina has a different thawing protocol.

By the way, you should send this question to Illumina. I'm sure they are asked it all the time. Their tech support is excellent.

--
Phillip

[ScottC]

I'm not sure what the purple color comes from

As an aside... I found it interesting that the V2 and V3 miseq reagents are different colours.

[xrobertdevine]

I asked this question "Life Tech does not like Ampliseq on the Miseq" because the previous person commented that. So, do you think it is not an issue? Can we actually use Ampliseq for target enrichment and run it on MiSeq?
Also, when you say life tech has their own sequencer- Are you talking about the Ion torrent sequencer?
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