Library quantification

[sbsuser]

is this abt the in lane Phix experiment?

[simonandrews]

How can you mix another library in to phix to get accurate quantitation? Can you explain.

Yes, just mix in the other library prior to putting it on the flowcell. After the run extract any reads from that lane which didn't map to PhiX and those will mostly come from your other library. The PhiX stats don't get messed up (other than %mapping) and you still have enough diversity to do the phasing calculations.

If you get around 10% of the other library from your lane we find that around 90% of the unmapped sequences come from this library. You can filter out most of the other PhiX reads by remapping against PhiX with relaxed constraints if you want, and you can get a pretty good idea of what's going on in your other library.

[sbsuser]

How can you mix another library in to phix to get accurate quantitation? Can you explain.

[simonandrews]

If you're running a control lane on your flowcells you can also mix (a best guess) 10% of another library in with PhiX to get a pretty accurate quantitation and also check on the quality of the library.

[sbsuser]

yes, deblock FC after 6 cyc and store in fridge. You can reuse the flowcell if you want to validate your GA.

[suludana]

Do you mean spend a Flow Cell to determine the exact density?

[sbsuser]

if you still cant make it from qPCR conc to get the right density of flowcell. i prefer you to sequence for 6 cyc on GA and make the flowcell with the right conc.

[suludana]

Hi,
I am using qPCR and Bioanalyzer to quantify my libraries before sequencing in a GAII. Even so I still can not get it right. Each run is a surprise (not always good). Does anyone have a really good method?
Thanks