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-   -   MiSeq Reagent Kit v3 (http://seqanswers.com/forums/showthread.php?t=33824)

paolo.kunder 09-17-2013 11:59 PM

MiSeq Reagent Kit v3
 
Hello!
Has anyone tried to perform RNA-Seq and ChIP-Seq with the new MiSeq Reagent Kit v3?
What about the throughput?
Any feedback?
Thanks
Paolo

MadsAlbertsen 09-18-2013 10:12 AM

We just did some stranded RNAseq libraries with MiSeq v3 kits.

Worked perfectly. 1340 k/mm^2 density and 32M reads (29M passed).

rgds
Mads

yaximik 09-21-2013 07:04 AM

Quote:

Originally Posted by MadsAlbertsen (Post 116657)
We just did some stranded RNAseq libraries with MiSeq v3 kits.

Worked perfectly. 1340 k/mm^2 density and 32M reads (29M passed).

rgds
Mads

I found 2 protocols on library loading, which differ greatly in load ranges. One starts from 10 nM libraries and provides dilution tables from 20 pM to 50 pM final library concentrations - without giving any hints at least what to start from to get 1200-1400 cluster density. Another manual starts from 4 nM library and provides dilution tables up to 20 pM final library concentration - again without a clue what to use to get to the new higher density. Which is correct manual?
My libraries are made from fragmented DNA, so I started from 10 nM library and diluted to final 25 pM according to the first protocol. I got only ~930 density. So, the entire range in the second protocol would lead to underutilization of v3 capabilities. Any comments from other experiences?

yaximik 09-21-2013 06:09 PM

All right, I did another run with the same library using the first protocol, but loaded the flow cell at ~33 pM instead of 25 pM as in the previous run. I got density up to 1144 with 85.2% PF as compared to density of 928 with PF 88% at 25 pM library load. Looks like the protocol 1 is the way to go.

MadsAlbertsen 09-22-2013 11:02 PM

Quote:

Originally Posted by yaximik (Post 116877)
All right, I did another run with the same library using the first protocol, but loaded the flow cell at ~33 pM instead of 25 pM as in the previous run. I got density up to 1144 with 85.2% PF as compared to density of 928 with PF 88% at 25 pM library load. Looks like the protocol 1 is the way to go.

Nice to see that you got it to work. We use the new loading reccomendations of 20 pM. However it is very difficult to estimate the right concentration of the samples due to the differences in insert length although we run all samples on a TapeStation. We are usually happy if we hit +/- 20 % of the density we aimed for.

rgds
Mads


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