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Old 12-28-2016, 07:05 AM   #1
LUR
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Location: Milan

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Default low fluorescence intensities read 2, HiSeq 2000

hi everyone
I have a problem with PE module Hiseq 2000
Basically the instrument read well the read 1 and the read index, but when arrive to the read 2, the fluorescence intensities are very low and sometime with Q30 around 0 (RNAseq libraries read at 50 PE).
I have opened a case with Illumina 2 month ago but I haven't the solution yet.
Someone have had the same problem?

Thanks a lot
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Old 01-03-2017, 11:37 AM   #2
pmiguel
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Well, unless you are using crazy old single read Illumina adapters, it sounds like something went awry during turn-around.
If you are under a service contract you should ask Illumina to replace the reagents so you can give it another shot.

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Phillip
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Old 01-04-2017, 05:54 AM   #3
helskel
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Default Have you checked your NaOH?

It is also possible that you have an issue with the NaOH. If the concentration of your NaOH in the PE module is off, you will not get through turnaround efficiently, and that can lead to all of the issues that you described.
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Old 05-05-2017, 01:15 AM   #4
LUR
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The problem was with the wash and mantainence wash on HiSeq. Probably a contamination with something but we don't know the origin of this contamination. We have solved the problem restored the mantainance wash with NAOH 0.1M. Now the instrument work well.
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Old 05-05-2017, 01:29 PM   #5
misterc
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If you switch back to the 0.5% Tween + 0.03% Proclin maintenance wash solution, be sure and rinse the NaOH from your lines. For good measure, I'd do two complete "maintenance wash" runs with just de-ionized water loaded in all reagent positions. This makes sure you don't get some nasty interaction between NaOH and the Tween/Proclin mix. The Tween/Proclin maintenance wash solution I've had much better luck with preventing clogs and such than with 0.1M NaOH, but it has the drawback of creating possible biofilms in the lines since Tween supports microbial growth if the Proclin doesn't sufficiently prevent it. Switching from Tween/Proclin to the 0.1M NaOH wash would also be best advised with a couple long water washes to rinse out the former.
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fluorescence, hiseq 2000, low intensity, paired end reads

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