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Old 12-23-2009, 03:02 AM   #1
hanjaylee
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Default the minimal amount of DNA required for deep-sequencing platforms!

hey. everyone! i have a sample containning many kinds of different cDNA, and i want to know their sequence and abundance in the sample. the problem is that the amount of the cDNA in the sample is limited. and i don't want to amplify them as this might introduce PCR bias, thus altering their relative abundance. can anyone tell me the minimal amount of DNA required for the next-generation sequencing? thanks a lot!

hanjay
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Old 12-25-2009, 10:17 AM   #2
pmiguel
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Quote:
Originally Posted by hanjaylee View Post
hey. everyone! i have a sample containning many kinds of different cDNA, and i want to know their sequence and abundance in the sample. the problem is that the amount of the cDNA in the sample is limited. and i don't want to amplify them as this might introduce PCR bias, thus altering their relative abundance. can anyone tell me the minimal amount of DNA required for the next-generation sequencing? thanks a lot!

hanjay
Most Next gen platforms will ask for pre-e(m)PCR to be done on a library for one reason or another.

The new 454 (GS-FLX) Rapid cDNA protocol, does not and asks for, I think, >200ng of RNA going into a library to make cDNA.

Protocols are available at 454.com/my454 --but you have to register first and this can take several days.

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Phillip
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Old 12-25-2009, 05:15 PM   #3
hanjaylee
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Originally Posted by pmiguel View Post
Most Next gen platforms will ask for pre-e(m)PCR to be done on a library for one reason or another.

The new 454 (GS-FLX) Rapid cDNA protocol, does not and asks for, I think, >200ng of RNA going into a library to make cDNA.

Protocols are available at 454.com/my454 --but you have to register first and this can take several days.

--
Phillip
thanks very much, Phillip! i will check the website. initially i was thinking of solexa, but it requires 5-10ug DNA, quite exceeding my sample.
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Old 12-26-2009, 01:42 PM   #4
shurjo
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Hi hanjaylee,

If you are using cDNA, the Illumina 10ug requirement should not be a problem for you. I routinely make libraries with whatever cDNA comes out of reverse transcribing 100ng of mRNA, so the amounts you mention should be totally usable. As regards amplification bias, the Illumina RNA-Seq protocol does have a final amplification step, so that may rule it out for you anyhow, but I would not worry about the amount of input cDNA.
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Old 12-26-2009, 02:03 PM   #5
pmiguel
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Hi hanjaylee,

If you are using cDNA, the Illumina 10ug requirement should not be a problem for you. I routinely make libraries with whatever cDNA comes out of reverse transcribing 100ng of mRNA, so the amounts you mention should be totally usable. As regards amplification bias, the Illumina RNA-Seq protocol does have a final amplification step, so that may rule it out for you anyhow, but I would not worry about the amount of input cDNA.
And, just to keep beating the "where does all the DNA go" drum:

rule of thumb: 1 ng of 1 kb DNA ~= 1 billion molecules.

That is double stranded DNA, so 100 ng of mRNA is about 200 billion molecules (if the RNAs are 1000 bases on average.)

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Old 12-26-2009, 05:09 PM   #6
hanjaylee
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thanks shurjo,
i am wondering what you use to RT, oligo-dT or gene-specific primers, to obtaion your cdna product!
hanjay
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Old 12-26-2009, 08:35 PM   #7
shurjo
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Actually, I use random hexamer primers.

Best,

Shurjo.

Last edited by shurjo; 12-26-2009 at 09:46 PM.
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Old 12-27-2009, 03:27 AM   #8
seqAll
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Hi Shurjo,

I would appreciate if you can guide me to some protocols using hexamer primers, or share your protocol. I am quite new to this field.

Best,
seqAll
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Old 12-27-2009, 10:38 AM   #9
shurjo
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You can download a copy of the Illumina protocol here:

http://tucf.org/htseq_mRNA-Seq_Sampl...04898_RevA.pdf

Regards,

Shurjo
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Old 01-13-2010, 02:43 PM   #10
kainsteven
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We have recently introduced a kit that requires as little as 500 pg of total RNA input, and produces ds cDNA compatible with the GAIIx workflow. Information about the product, including the User Guide (protocol) can be found here: http://www.nugeninc.com/nugen/index....na-seq-system/

Best,
Steve
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Old 01-14-2010, 07:36 AM   #11
GW_OK
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Theoretically, for the Illumina, you only need 2 ul of a 10nM prepped library to go into the cluster station/cBot. You might be able to get there without using PCR, if you used Sangers PCR-less library prep.
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Old 01-14-2010, 04:18 PM   #12
hanjaylee
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GW OK,
THANKS FOR YOUR REPLY! would you please give me some more detailed information, as this is very important for me. thanks a million!!
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Old 01-15-2010, 08:40 AM   #13
GW_OK
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As it stands the Illumina adapters come in two parts: the part that is ligated, containing the sequencing primer binding site, and the part that is added during the amplification PCR, containing the flowcell binding site. Basically what you need to do is have oligos synthesized that containing both of these sites. You can find the sequences in another thread on the top of the Illumina sub-forum. Since these oligos have both the flowcell binding site and the sequencing primer binding site you don't need to do an amplification step. This was mentioned in Sangers paper "A large genome centre’s improvements to the Illumina sequencing system"
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Old 01-15-2010, 05:32 PM   #14
hanjaylee
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very much thanks, i will check it out.
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Old 02-23-2010, 12:41 AM   #15
staoudi
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Quote:
Originally Posted by kainsteven View Post
We have recently introduced a kit that requires as little as 500 pg of total RNA input, and produces ds cDNA compatible with the GAIIx workflow. Information about the product, including the User Guide (protocol) can be found here: http://www.nugeninc.com/nugen/index....na-seq-system/

Best,
Steve
Hi,
I am interesting in using the Ovation RNA-Seq system. I
have a few questions:

1. Is the system compatible with bead purified mRNA as a starting point?

2. The product literature discourages the use of yeast tRNA or glycogen as
carriers when extracting RNA, are there any alternative carriers that you
can recommend using?
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Old 07-30-2010, 10:21 AM   #16
kainsteven
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Default Ovation RNA-Seq System

Hi Staoudi,

In answer to your questions:

1. Yes, our system is compatible with purified mRNA. We have not characterized the input amounts for mRNA, as our system was validated for total RNA in the range of 500 pg to 100 ng. In theory it should be less, but I'd start in the 5-10 ng range if you have this much material.

2. We do not recommend tRNA as a carrier as this can be efficiently amplified in the Ribo-SPIA process used with the Ovation RNA-Seq System. In addition, glycogen is known to inhibit reverse transcription, so we do not recommend this carrier. We have not tested it extensively with our system, but a possibility is the inert carrier linear polyacrylamide (LPA). The addition of LPA to a nucleic acid precipitation can enhance recovery of small amounts of DNA or RNA.

Best,
Steve
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