Currently there is an issue with the quality of the reagents specifically with the 600V3 cartridges. They cannot seem to identify the cause.
For the last 150 cycles on the reverse strand you will see a massive drop off in Q30 and increase in error rate.
This drop in the reverse strand also appears in the V2 kit (2*250). Diluting the clusters to 600-700 / mm2 helps to improve the run stats.
Hello,
There is a global chemical trouble with the 2x300 kits of Illumina MiSeq. It affects the quality of read 2 and It is worse on the final 100 flows and this fact doesn't allow an overlap between the first and second read because the sequence isn't long enough to overlap.
Does anyone still experienced quality problem with the Illumina v3 600 kit, read 2? I had very bad quality in the read 2 in my last run.
I have also obtained very bad quality read2 (completely unusable). As I resequenced the same set of libs using the same primers and they yielded good quality r2, I think that it was a problem with the kit.
Has anyone succeeded in getting the kit back from Illumina? In my case the TS claimed the problem was due to my failing to add r2 custom primer (and I'm sure it was not the case)....
Yes, we ve got a replacement kit, by showing some result slides from the bad run and slides from a previous good run.
hi guys,
one question, how do you quantify each library and then pool libraries. What is the best way to do this? thanks so much.
What I'm doing is each library is quantify by Quibit and Bionalizer (to see the size mean) then, each library is diluted to get 4nM and take 2ul of each one to make a pool at 4nM. after that I check the quantity by qubit and bionalizer again to make some correction a get real 4nM of pool. Finally, I take 5ul of pool to diluted at 20pM and run the sequences. my cluster density was 849 instead of 1200-1400 according manual instruction for 600v3 (2x300)
Hello everybody,
Has any of you seen quality of reverse Illumina reads similar to what I attach bellow? This was v1v3 region. What could be the reason there are around 25% of all sequences that have the strange appearance? Thanks. Stanislav.
Is there an update on this (Sept 2019) - 2015-2017 (and maybe into early 2018), the verdict was still that the v2 chemistry was better compared to the v3.
I'm submitting samples for MiSeq sequencing and may only have the v3 chemistry option. Thoughts? updates?
We just experienced similar problems, one run got 50% Q30 and the other did not produce any output. The first using a v3 and the second a v2. With 550 bp fragments.
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