Low Quality Reverse read Miseq

[valeriagomez99]

Hello every one

i work with Miseq illumina using V2 chemistry (500 cycles) and in the last runs i´ve had a problem with the quality of the reverse read; the forward read has a Q30 <90%, but the reverse decrease at point of 12% on mi last run. Has anyone idea of the possible reason?

This was mi last run, the cartridge is for 500 cycles but only i used 181 and 181

[Ben3]

valeriagomez99 has this happened more than once? I can't understand why the quality would drop so low. Also, what kind of samples were you running?

[valeriagomez99]

This have happened several times. The samples for this run were microsatellites, but 16s and ITS amplicons have had the same problem. i used expired cartridge, but if this were the problem i think that forward read quality also would be affected

[Ben3]

valeriagomez99 yeah that doesn't sound good. Have you spoken to Illumina about this? I'm going to assume it's something wrong with the MiSeq because clearly, something is going wrong right before or during read 2. And I've used tons of expired cartridges and had no issues, unless they were accidentally thawed and refrozen.

[valeriagomez99]

Ben3, yes the illumina personal checked the Miseq and all is well, and between the failed runs we have used new cartridges and the sequencing finished fine with a good reverse, this only happens with expired cartridges which makes us think it may be the cause, the cartridge always remains frozen in any case.