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?
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?
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