HiSeq2000/ cBot poll - input vs pf

[Newie]

Every instrument/ library is same, same but different...

Curious what concentration people are loading per lane vs pf cluster densities they are getting on their cBot/ HiSeq instruments (indicating library type and size distribution would be great)?

We are planning on sending our libraries out for HiSequencing or generating fc's in house and sending them out - curious to know what to expect?

Thanks for your help!

[avadhanam]

We use 8pM as the final concentration that goes into the flow cell. Although illumina recommends 10pM, we got good coverage for 8pM too. With this you get a cluster density of 400-600.
RNA libraries of size 320bp or so is the final size verified using bioanalyzer. Hope this info helps.

[pmiguel]

For the current run we used 16 pM as measured by the KAPA qPCR kit, using the KAPA standards. We do correct the concentration for the average size of each library.
This puts us at 800-850 K clusters/mm^2. This gave us 92-95% pass filter for clusters.

We had problems with both cluster density and getting even numbers of clusters/library but I think we have worked through them. What we changed:

(1) Tightened up the size distributions of our libraries. We now do "upper cut" ampures to remove higher molecular weight products.

(2) Stopped trying to use phiX as the qPCR standard and switched entirely to the KAPA protocol. (Which also involved using much higher dilutions of the initial samples.)

(3) Now we take "heroic measures" to mix samples during serial dilutions. 10 full volume pumps of the solution may not have been enough. Chase that with vortex mixing.

If this stays stable we may move our cluster density up. The instrument appears to produce excellent results at 1000 K clusters/mm^2, so we could take advantage of that -- if we were confident we would not come in 50% high...

--
Phillip

[mama]

upper cut

Phillip, can you explain me in which step during the library prep do you do the "upper cut" ampures to remove the higher molecular weight product?
Did you try to do it in the chipSeq library prep?

[GW_OK]

We load 18pM, shooting for 850-950K/mm^2 with ~90% PF. It does vary a bit, usually upwards, but as long as the cpmm2 is below ~1200K it usually works out with >80% PF. You have to be careful, though, as there's a steep PF drop lurking immediately above that.

We also do the dual size selection with Ampure beads, post-PCR, and use the KAPA kit and included standards. And I've also noticed that mixing properly is key to a good qPCR.

[pmiguel]

Phillip, can you explain me in which step during the library prep do you do the "upper cut" ampures to remove the higher molecular weight product?
Did you try to do it in the chipSeq library prep?

I have not tried upper molecular weight cuts for chipseq. The "upper cut" could be done before or after enrichment PCR.

--
Phillip

[huguesparri]

7 pM for DNA (gDNa and ChIpseq) application.
8 pM for RNAseq application.
We are aiming 750 to 850 thousand cluster/mm².
Libraries are quantified using Kapa kit.

[pmiguel]

7 pM for DNA (gDNa and ChIpseq) application.
8 pM for RNAseq application.
We are aiming 750 to 850 thousand cluster/mm².
Libraries are quantified using Kapa kit.

What instrument is this?

--
Phillip

[huguesparri]

Oups, sorry: we are using a HiSeq2000 and v3 chemistry.

[pmiguel]

I was asking because your numbers seem so far away from what we see. Could that be 17/18 pM you use, instead of 7/8 pM?

Or are you libraries much shorter than the KAPA standard and thus, effectively, of higher concentration than what the KAPA kit tells you?

--
Phillip

[huguesparri]

Nope, it's really 7 and 8 pM.
When we quantify a library using the Kapa kit, we take into account the length of our fragments.
For RNAseq, we define a "region" on the bioanalyzer and use the "average length" calculated by the bioanalyzer.
For gDNA, it's more simple as there is only one sharp peak.

[genomeseeker]

We routinely use 11 and 12pM on HiSeq2000 and generally get densities in the 650-850K/mm2 range. We quantify with the KAPA kit, as well, using their standards and adjusting concentration for avg library size.