the minimal amount of DNA required for deep-sequencing platforms!

[hanjaylee]

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

[pmiguel]

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.

--
Phillip

[hanjaylee]

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.

[shurjo]

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.

[pmiguel]

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.)

--
Phillip

[hanjaylee]

thanks shurjo,
i am wondering what you use to RT, oligo-dT or gene-specific primers, to obtaion your cdna product!
hanjay

[shurjo]

Actually, I use random hexamer primers.

Best,

Shurjo.

[seqAll]

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

[shurjo]

You can download a copy of the Illumina protocol here:

http://tucf.org/htseq_mRNA-Seq_SamplePrep_1004898_RevA.pdf

Regards,

Shurjo

[kainsteven]

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

[GW_OK]

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.

[hanjaylee]

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!!

[GW_OK]

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"

[hanjaylee]

very much thanks, i will check it out.

[staoudi]

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?