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-   -   10, 50X human exomes per flowcell (http://seqanswers.com/forums/showthread.php?t=17632)

JPC 02-13-2012 07:59 AM

10, 50X human exomes per flowcell
 
Hello All, just in case anyone in interested.....we are able to get 10 exomes at 50X coverage from a single flowcell (with the standard 1um microbeads).

We aren't getting there every time, we count beads on the nanodrop, we think we've got 400M per lane but the lanes are often quite pale after loading.....we haven't figured out what's going wrong here yet?

When it works well, one lane gives us 180 - 200M reads, 18% don't map (using LifeScope and ECC).

Is anyone doing considerably better?....would you recommend the nanobeads to improve our figures?
Is anyone doing worse? I'm happy to share what I have learned!

JPC

jimmyinfrance 02-27-2012 05:40 AM

HI JPC,

10 exomes/flowchip sounds good (one E120?), are you doing forward and reverse reads? What exome capture are you using?

JPC 02-27-2012 06:23 AM

Yes it's one E120. Our capture is with Agilent SureSelect (version 4) and we mainly use forward reads with ECC module (and there for no reverse reads).

We haven't tried nanobeads yet and I'm not sure it will be worthwhile if Wildfire is released on time, have you tried them?

jimmyinfrance 02-28-2012 01:11 AM

Thanks, gather your happy with the ECC? What read length are you using? We are using barcoding prior to capture (TargetSeq mostly), cost effective for the capture step but more off target reads, so our seq footprint is bigger, not sure that we will reach 10 exomes/flowschip as a result.

We haven't tried nanobeads, and Wildfire looks/seems a more interesting way of increasing capacity (at least according to what Lifetech say Wildfire can do...I'd like to see it in action!). July 2012 release for Wildfire according to Lifetech here.

JPC 02-28-2012 01:22 AM

Yes we are very happy with the ECC. It basically improves the quality of a proportion of the reads that would other wise be discarded so we get a higher return per flowcell.

We haven't tried the TargetSeq but my understanding is that it's a straight rebrand of the Roche kits, we may take a look at it in the summer if the cost is significantly lower.

jimmyinfrance 02-28-2012 01:35 AM

That Targetseq = Nimblegene is our (limited!) experience. We tried both and found Targetseq outperformed (just), with slightly higher on target reads, and there a buffer that facilitates the hybridisation process when one includes adaptors/barcodes prior to the capture (in addition to barcode "blocking" oligos). What that buffer is I don't know (I would like to!)

I gather from Agilent they are developing something similar, their kit promises multiplexing of 8 - 16 exomes prior to capture. They (agilent) tell me that ontarget reads remain high. Any experience?

JPC 02-28-2012 01:37 AM

Not yet, but I've been told the same from our rep so we're looking forward to trying it.

JPC 06-26-2012 02:21 AM

We're now getting 12 exomes from a single flowcell with an average coverage of 57 fold (from a single E120). That's 780M beads raw data.

We're still struggling with pooling our completed libraries though. In our latest run we have 1 sample with 180X and 4 samples under 50X. We are using qPCR to quantify, then doing serial dilutions to create the input DNA. Is there anything else I can do to get a proper balance?

JPC 07-10-2012 04:05 AM

Further to my last post I should point out that we can see that the qPCR concentrations that we have been getting do not show any relationship to the bead counts we see on the 5500. The concentrations we see on our bioanalyser do correlate with our bead counts so we will be pooling based on what our bioanalyser tells is from now on. I've started a new thread on this topic "Pooling for emPCR" which I will update after my next run.
JPC


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