Ah, not what I meant. I have the P5 and P7 oligos and use them for KAPA (SYBR green) qPCR. I thought you meant the fluorescent reporter probe method. I thought that was what "real time" specified. I guess not.
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Phillip
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The primers are the standard flowcell sequences. We actually now use the Kapa Biosystems kit. We buy it in very large bulk and the cost per library is very good. Better than building our own kit and standards.
We're working on a probe-based assay for a couple of unique situations that isn't ready for sharing yet, but it will still use the P5 and P7 flanking primers. They are also the same sequences that are used in the Nimblegen kit for amplifying the final libraries.
Can those sequences be "legally" posted here? Happy to post them if it isn't going to cause anyone to have issues.
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Originally posted by csquared View PostFor library pooling, you will absolutely see the best results with real-time PCR quantitation prior to pooling.
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Phillip
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Excellent, many thanks for all the advice, I think I have a good plan of action now.
@huguesparri: we are using a commercial facility who do indeed use PhiX controls. I pay for a per lane service so whilst I am absorbing their costs of 'losing' one lane I can be confident in the run.
@protist: as I am using the Truseq v2 kit I had previously used the 'resuspension buffer' as the diluting agent. I had a nightmare previously when dilutions from Qubit were WAY out (>2x) and the facility did not check them pre-run, so ended up with a single-end run due to too high a cluster density. I am thus checking around for all other options.
@csquared: as above, I may well use qPCR as this hasn't been done previously in our lab and I am keen to try it.
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For library pooling, you will absolutely see the best results with real-time PCR quantitation prior to pooling. Of course it is possible to do well with a size estimation using bioanalyzer or caliper or Advanced Analytical and DNA quantitation with a fluorescent means (Qbit, picogreen, etc). Spectrophotometry would be last on my list of recommendations.
The key is pooling in molar equivalents and pooling libraries of reasonably similar insert sizes. The means to that end can be quite variable.
Protist and others have great suggestions.
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Originally posted by bruce01 View PostThanks for the reply. I am leaning towards the 24 per lane owing to the final point you make. For reference what are you using to quantitate libraries? I have used Qubit and Nanodrop but am sceptical that they work particularly well and am keen to get good equimolar pools made to send for sequencing.
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I hope I'm not about to say something stupid but do you plan to load samples on every lane?
We definitely keep the 4th lane for a control (PhiX). And as RNAseq sequences are unbalanced in their first bases (it seems to be due to the random priming during reverse transcription), it might be wise to keep a PhiX on lane 5.
And for the quantification, you can use qPCR. Don't use Agilent for quantifying. Just use it to check your library quality.Last edited by huguesparri; 08-27-2012, 04:55 AM.
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Thanks for the reply. I am leaning towards the 24 per lane owing to the final point you make. For reference what are you using to quantitate libraries? I have used Qubit and Nanodrop but am sceptical that they work particularly well and am keen to get good equimolar pools made to send for sequencing.
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Best choice will depend on how accurately you pool the libraries in molar equivalents and if all libraries sequence with equal efficiency.
From a sequencing standpoint the two choices are pretty much equal. Informatics will be easier with 1 lane per samples as you wont need to demultiplex and then combine lanes to gather the full reads for a single sample. A small step but still a step that needs to be completed in option 1.
For what its worth, we typically use both options. For whole genome sequencing, we are pooling anywhere from 12 to 96 samples and running that pool over many lanes. Once a good pool is made, its easier to sequence that pool lots of times than many smaller pools.
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Multiplex scheme for Hiseq run
Hi,
I have an issue I need to resolve regarding multiplexing libraries for a Hiseq run.
There are 96 libraries, I am making them with Truseq V2 RNA prep kits which have 24 available barcodes. So Each barcode will be used 4 times. I have 8 lanes in which to run my libraries. Am I better off:
(i) multiplexing 24 per lane and having essentially 2 technical replicates per library or
(ii) multiplexing 12 per lane and having 1 technical replicate.
My problem is that whilst scheme (i) might reduce impact of variation in number of reads per lane, having more libraries multiplexed may also increase the disparity (variance) between numbers of reads per library.
What other reasons are pro/con each of these schemes? Your thoughts are greatly appreciated. I did try and search the forum and keep an eye out for this as I knew I would be doing this prep for the last few months but haven't seen discussion. Pointers to relevant info also appreciated
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