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Old 03-16-2010, 12:58 AM   #1
ezilybored
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Default Bioanalyser results???

Hi there everyone,

I'm performing some chip-seq and have just ligated the adaptors to my product. I sent it to another group with a bioanalyser to test and have just got the data back but it doesnt look too good I fear.
I have a large peak at around 70bp which I am guessing is adaptor carryover. However in a few samples there is another peak at around 125 bp. Is this another adaptor peak or is this my library? I would like to know before i continue and atempt to size select out the 70bp peak.
The bioanalsyer data is attached.
Many thanks.

Ben
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File Type: pdf 12.3.10_2010-03-12_14-52-10.pdf (971.4 KB, 192 views)
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Old 03-16-2010, 04:09 AM   #2
pmiguel
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Originally Posted by ezilybored View Post
Hi there everyone,

I'm performing some chip-seq and have just ligated the adaptors to my product. I sent it to another group with a bioanalyser to test and have just got the data back but it doesnt look too good I fear.
I have a large peak at around 70bp which I am guessing is adaptor carryover. However in a few samples there is another peak at around 125 bp. Is this another adaptor peak or is this my library? I would like to know before i continue and atempt to size select out the 70bp peak.
The bioanalsyer data is attached.
Many thanks.

Ben
Hi Ben,

A couple of points:

(1) What type of sequencer are you making this library for? (The adaptors you ligate on will be various sizes, depending on the platform you intend to use.
(2) Could you provide some other image format than pdf? Given the recent spate of browser-embedded acrobat issues, it really is not safe to open a pdf from an unknown source. I mean, you write like one of us, but so might a skilled phisher.

--
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Old 03-16-2010, 04:18 AM   #3
ezilybored
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Hi there sure I'll upload it in a different file format now.

We've prepared the library for use on an illumina machine using the illumina adaptor ligation kit following the protocol as outlined in the handbook.
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Old 03-16-2010, 05:40 AM   #4
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Originally Posted by ezilybored View Post
Hi there sure I'll upload it in a different file format now.

We've prepared the library for use on an illumina machine using the illumina adaptor ligation kit following the protocol as outlined in the handbook.
I don't have much solexa background. I do see that spike around 120 bases in 3 of your samples. Were you going to do a gel-based size selection? If so, maybe you could exclude that peak then?

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Old 03-16-2010, 05:43 AM   #5
ezilybored
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Hi,

yes that band does appear. I was planning on size selecting again to remove that and the lower 70bp band. However the lack of a band around 200bp is worrying. There is a slight darkening of the gel there, or maybe i am looking overly hopefully, is this enough to run on the machine?

Ben
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Old 03-16-2010, 06:29 AM   #6
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Hi,

yes that band does appear. I was planning on size selecting again to remove that and the lower 70bp band. However the lack of a band around 200bp is worrying. There is a slight darkening of the gel there, or maybe i am looking overly hopefully, is this enough to run on the machine?

Ben
You will want a Solexa user to answer that.

By the way, the new Agilent "High Sensitivity" DNA chips are around 100x more sensitive, so assaying your libraries on one of them would be better. Even with the data you have, your bulge is being flatened down to base line by the high relative signal from the standards. If you have the .xad file you could zoom in on the putative bulge area, and do a smear assay to attempt to roughly quantify the amount of library you have.

I do think you problem could just be sensitivity. Because 1 ng of a 200 base fragment is 5 billion molecules. But with this chip distinguishing 1 ng sample from baseline would be difficult.

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Old 03-16-2010, 08:02 AM   #7
ezilybored
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You will want a Solexa user to answer that.

By the way, the new Agilent "High Sensitivity" DNA chips are around 100x more sensitive, so assaying your libraries on one of them would be better. Even with the data you have, your bulge is being flatened down to base line by the high relative signal from the standards. If you have the .xad file you could zoom in on the putative bulge area, and do a smear assay to attempt to roughly quantify the amount of library you have.

I do think you problem could just be sensitivity. Because 1 ng of a 200 base fragment is 5 billion molecules. But with this chip distinguishing 1 ng sample from baseline would be difficult.

--
Phillip
Hi,

yes, getting an answer from a solexa user would be quite helpfull. I have enquired with our neighbouring lab about whether it is possible to run the samples again on a high sensitivity DNA chip and also if they know how to perform a smear assay on the data. I am also contemplating re-attempting the ligation, this time using less a more dilute adaptor stock as I have read on here that 1:10 is still to concentrated. I will also try taking a slightly higher slice of the gel, from 200-300 bp this time, as initially i believe i went a bit below 200 bp as the illumina protocol described taking +/- 25bp either side of 200 bp.

Ben
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Old 03-16-2010, 11:32 AM   #8
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Hi, maybe this can help a bit.
As I recall, Illumina only recommends checking the library on the bioanalyzer after the final enrichment/PCR step. Perhaps you should not expect to see anything at this stage - too low concentration? Their mRNA-seq sample prep protocol has a good image of how a bionanalyzer trace is supposed to look after the final PCR step - check the last section "Validate the library".
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Old 03-17-2010, 12:53 AM   #9
ezilybored
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Originally Posted by larissa View Post
Hi, maybe this can help a bit.
As I recall, Illumina only recommends checking the library on the bioanalyzer after the final enrichment/PCR step. Perhaps you should not expect to see anything at this stage - too low concentration? Their mRNA-seq sample prep protocol has a good image of how a bionanalyzer trace is supposed to look after the final PCR step - check the last section "Validate the library".
Hey there,

unfortunately this run was performed after the final pcr enrichment step and so i would have thought we may see something.
will check that mRNA-seq protocol now, thanks.

Ben
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Old 03-17-2010, 04:24 AM   #10
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Originally Posted by ezilybored View Post
Hey there,

unfortunately this run was performed after the final pcr enrichment step and so i would have thought we may see something.
will check that mRNA-seq protocol now, thanks.

Ben
But, again, if your bioanalyzer chip is not sensitive enough you could have less library than the example in the manual, but plenty to sequence. That is why the high sensitivity chips are so useful. You can assay the quality of a library's size distribution without having to do extra PCR which could bias the library.

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Old 03-17-2010, 04:31 AM   #11
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Originally Posted by pmiguel View Post
But, again, if your bioanalyzer chip is not sensitive enough you could have less library than the example in the manual, but plenty to sequence. That is why the high sensitivity chips are so useful. You can assay the quality of a library's size distribution without having to do extra PCR which could bias the library.

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Hi Phillip,

I am currently in conversation with my neighbour who owns the bioanalyser about purchasing some of these higher sensitivity chips. Thank you very much for bringing them to my attention.

Ben
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Old 03-17-2010, 08:38 AM   #12
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Hi Ben and Phillip,
I just came across a review on Current Protocols in Molecular Biology 2010 (RNA-Seq: A Method for Comprehensive Transcriptome Analysis by Nagalakshmi et al). This is specific for the Illumina platform, so you may find it useful. After the cDNA library is generated, they recommended checking on a NanoDrop and specifically mention "The ideal concentration is 15 to 25 ng/μl. If the cDNA concentration is lower, the
sequencing eficiency will be low." Check page 4.11.7.
Phillip, I know your experience is mainly with the Solid platform, in that case, which is the minimum amount/concentration of the final library that generate a reasonably good run with the ABI machine? Just curious Also, thanks for bringing up the high sensitivity chips. I was also not aware Agilent had that one.
Thanks!
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Old 03-17-2010, 08:49 AM   #13
ezilybored
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Quote:
Originally Posted by larissa View Post
Hi Ben and Phillip,
I just came across a review on Current Protocols in Molecular Biology 2010 (RNA-Seq: A Method for Comprehensive Transcriptome Analysis by Nagalakshmi et al). This is specific for the Illumina platform, so you may find it useful. After the cDNA library is generated, they recommended checking on a NanoDrop and specifically mention "The ideal concentration is 15 to 25 ng/μl. If the cDNA concentration is lower, the
sequencing eficiency will be low." Check page 4.11.7.
Phillip, I know your experience is mainly with the Solid platform, in that case, which is the minimum amount/concentration of the final library that generate a reasonably good run with the ABI machine? Just curious Also, thanks for bringing up the high sensitivity chips. I was also not aware Agilent had that one.
Thanks!
Hi Larissa,

do you know if the same concentrations apply for ChIP-seq?

Ben
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Old 03-17-2010, 09:11 AM   #14
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Do not know, sorry... Have you tried Illumina tech support? I have had good experiences in the past with them.
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Old 03-17-2010, 10:04 AM   #15
pmiguel
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Quote:
Originally Posted by larissa View Post
Hi Ben and Phillip,
I just came across a review on Current Protocols in Molecular Biology 2010 (RNA-Seq: A Method for Comprehensive Transcriptome Analysis by Nagalakshmi et al). This is specific for the Illumina platform, so you may find it useful. After the cDNA library is generated, they recommended checking on a NanoDrop and specifically mention "The ideal concentration is 15 to 25 ng/μl. If the cDNA concentration is lower, the
sequencing eficiency will be low." Check page 4.11.7.
Phillip, I know your experience is mainly with the Solid platform, in that case, which is the minimum amount/concentration of the final library that generate a reasonably good run with the ABI machine? Just curious Also, thanks for bringing up the high sensitivity chips. I was also not aware Agilent had that one.
Thanks!
Concentration itself is not much of an issue as the volume of library that can be added to the emulsion PCR is substantial. The DNA sequencing manager here was very happy with some libraries he ran on a high sensitivity Agilent chips yesterday and these were in 1 ng/ul range. Since that works out to 5 billion library (200 bp) molecules/ul, it should be plenty.

But we do qPCR to determine how much to add to the emulsion PCR.

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Old 03-17-2010, 12:47 PM   #16
james hadfield
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I do not think your library prep has worked.

Running these on a high-sensititvity kit will give you more confidence that they have not so it is probably still worth it for peace of mind.

Did you read the Sanger paper from 2008? This has lots of tips for improving library prep. Most of all for ChIPseq you need to titrate the adapters against input DNA, many of my users are using 50x lower adapter concentration.

Use teh gel picking tips from Cleaver Scientific or elsewhere to make sure you only get your band of interest into the final PCR. Gel purify before PCR but also cut bands (with new tips) at above and below your region of interst. If the PCR goes wrong then you have a sample to repeat it from, albeit at a different size.

Forget the nanodrop! Illumina have a QPCR protocol now try that.

Good luck.
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