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Old 06-09-2010, 01:30 AM   #1
jasmineja
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Default phi x control on the same lane with sample?

We would like to run RNA seq- we have 8 samples. Do you have any experience with running the phix control WITH one of the samples? Could we still use the phi x as the control this way?
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Old 06-09-2010, 06:37 AM   #2
Bruce E
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Default mRNA seq

We have been running RNA-seq with out a PhiX for over a year and it works fine. Giving up a lane for a standard is too expensive. The only reason you need a PhiX besides QC is if you are doing an unbalanced sample such as ChIP-seq, small RNA, Methyl-seq, or an unbalanced sequence. If you really want you can spike PhiX into your your sample at ~1% for QC but I do not believe this would help for phasing or matrix generation if your sample is unbalanced.
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Old 06-10-2010, 12:42 AM   #3
jasmineja
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Thanks for your reply, the possibility of running with no phix is appealing.could you specify the description of balanced or unbalanced samples,
thanks
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Old 06-10-2010, 06:15 AM   #4
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It's just that the GAII "calibrates" its matrix for the base calling step in the first cycles. In order to have a good calibration, the base abundancy in these cycles must be rather equal (25% A, 25% G, 25% C, 25% T, more or less).
Usually, you use PhiX as a reference balanced sample. But if you know that at least one of your samples is balanced, you can specify to the machine to use it as a reference.
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Old 06-10-2010, 01:58 PM   #5
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If you do run into issues with a run, Illumina is much more likely to replace your reagents if you have run the phiX control. Just a warning.
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Old 06-11-2010, 12:16 PM   #6
cbrennan
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As a core lab - we run a .04pM PhiX spike in each lane. Even if the clients library fails to produce clusters there is still enough to align PhiX & show that it was not an instrument problem. Bruce is correct it won't help with matrix/phasing etc if you have a biased sample you're trying to rescue.

The version of RTA/OLB you're using makes a huge difference, the newest RTA (1.6.47) handles RNA & CHiP much better than previous versions. If you do need to use OLB & a control lane to rescue samples - you don't have to use PhiX. Say you've got a good human genomic lane on that same flowcell -- you can use that as your control-lane. The only situation I've come across that you can't rescue is when the lane is just too over-concentrated - but a control lane wouldn't help in that situation either.
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Old 06-14-2010, 01:52 PM   #7
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Quote:
Originally Posted by cbrennan View Post
The version of RTA/OLB you're using makes a huge difference, the newest RTA (1.6.47) handles RNA & CHiP much better than previous versions.
I am working with ChiP and have GAPipeline 1.4.0. Can I get the newer version from Illumina ?
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Old 06-14-2010, 02:08 PM   #8
cbrennan
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Yes -- you need an iCOM account, I believe you need your FAS to set it up for you.

Go here:

https://icom.illumina.com/

Once it's set up there will be a download section -- look under software.

You can download OLB v1.6.1 and run basecalling from images -- but it sounds like you're perhaps still on the 1.44mm flowcells, so if you're going to use Gerald for alignment you may have to use Casava v1.5.1
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Old 06-15-2010, 08:36 AM   #9
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cbrennan,

Thank you very much for the information. Actually, we have been getting our sequencing done at various places so I am not sure of the flow cell size. I guess it is something I will have to pay more attention to.
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Old 06-16-2010, 01:15 AM   #10
jasmineja
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thanks for the idea of running 0.04 pM phiX in each lane.
I installed the newest RTA, anyway it is neede for v.4.so we are ready to run 8 samples instead of 7... we will try. thanks
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Old 07-21-2010, 11:01 AM   #11
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Quote:
Originally Posted by cbrennan View Post
As a core lab - we run a .04pM PhiX spike in each lane. Even if the clients library fails to produce clusters there is still enough to align PhiX & show that it was not an instrument problem. Bruce is correct it won't help with matrix/phasing etc if you have a biased sample you're trying to rescue.
When do you add your PhiX to the sample? I'm assuming if it's already at a pM concentration then it's after denaturation and you're adding PhiX diluted in HT1 to your samples that are denatured and diluted in HT1 as well.

Also we're multiplexing samples (running a control lane) but wanted to spike our samples with phix to help give the sample lanes more diversity. Does anyone have any experience with that? I'm wondering at what concentration to use. A little background, our barcodes were designed ourselves, we are not using Illumina's barcoding system.
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Old 07-30-2010, 01:00 PM   #12
btarrier
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Default PhiX Spiking

Quote:
Originally Posted by cmawhinney View Post
When do you add your PhiX to the sample? I'm assuming if it's already at a pM concentration then it's after denaturation and you're adding PhiX diluted in HT1 to your samples that are denatured and diluted in HT1 as well.

Also we're multiplexing samples (running a control lane) but wanted to spike our samples with phix to help give the sample lanes more diversity. Does anyone have any experience with that? I'm wondering at what concentration to use. A little background, our barcodes were designed ourselves, we are not using Illumina's barcoding system.
We add PhiX to our samples after denaturation during the final dilution. We denature PhiX and dilute to 4pM as you would any other sample. Instead of making the final sample dilution in 1ml, we make it up in 990ul and then add 10ul of 4pM PhiX. This gives you a final concentration of 0.04 pM PhiX. This is typically about 0.5% depending on the sample concentration that we are loading.

I don't think that this will help you at all with base composition in barcoded samples. The software is looking for a somewhat uniform base distribution and 0.5% will not help if you only have two (or three) out of four bases present on any given cycle. We also use our own barcodes and PhiX at that concentration is basically invisible compared to the samples present.
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Old 08-06-2010, 05:31 AM   #13
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I use the same sort of protocol as btarrier.
Denature sample and PhiX separately, add PhiX to sample. However, I use only 2uL of a 600pM PhiX (subtracting that 2uL from the HT1 volume added to the sample), giving 1% PhiX.
So far this has been working well.
We simply include a balanced sample in each run and set it as the control lane, thus avoiding sacrificing a lane to PhiX
We've never run a flowcell without an unbiased sample, as the addition of PhiX is not enough to normalize the base ratio and assure correcting calling.

Last edited by KorNor; 08-06-2010 at 06:54 AM.
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Old 08-12-2010, 09:33 AM   #14
cmawhinney
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Quote:
Originally Posted by btarrier View Post
I don't think that this will help you at all with base composition in barcoded samples. The software is looking for a somewhat uniform base distribution and 0.5% will not help if you only have two (or three) out of four bases present on any given cycle. We also use our own barcodes and PhiX at that concentration is basically invisible compared to the samples present.
We ended up spiking with 30% PhiX (at 7.5pM) because we realized spiking in such a low % of PhiX wouldn't help. Doesn't look like the 30% helped much either since Pipeline is having a hard time with base calling.

These samples were run with v2/3 reagents and thus run on version 1.5 of the Pipeline. I am now trying to run on OLB 1.6 as well as tweaking a lot of the parameters (for example setting it to use 10 bases, as opposed to the default of 4 in OLB 1.6) for cluster identification and it's not helping that much. FYI, We did run a separate control lane of PhiX as well.

If a anyone has any tricks for the analysis of barcoded samples! Since front indexing isn't an Illumina supported protocol they aren't much help.
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Old 08-12-2010, 10:42 AM   #15
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cmawhinney -- did you try running OLB on a few tiles out past the barcode to generate a crosstalk matrix, and then run a full pipeline from cycle 1 importing that matrix? That's worked for me in the past.
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Old 08-12-2010, 10:48 AM   #16
cmawhinney
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^ I have not but this was something that was just suggested to me and I might give it a try. For generating the matrix file, how many tiles do you think is adequate? And do you do all cycles or can I shorten that as well?
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Old 08-12-2010, 11:21 AM   #17
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I usually pick 10-20 tiles and then run for 25 cycles. I try to avoid picking tiles from the flowcell edges as they're more prone to have problems. It runs pretty fast and gives you a Bustard.xml that you can check it to see if the pass filter/etc looks reasonable before doing a full pipeline.
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Old 11-30-2011, 01:10 PM   #18
CriCru
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Does adding the PhiX spike-in to each sample have the same effect as adding an ERCC control? Ambion offers this ERCC control to help with accurately quantify transcript levels, but I'm not sure if I need to invest in this addition or not... Any thoughts?
Thanks
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