View Single Post
Old 11-20-2009, 08:11 AM   #17
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Quote:
Originally Posted by 454Sequencing View Post
Hi everyone,

If you've not heard, earlier this week we announced the availability of a new cDNA library preparation protocol. This is a straightforward, general utility transcriptome sequencing method, that takes advantage of an existing Roche ds cDNA synthesis kit and the newly released Rapid Library kit from 454.
[...]
Best regards,
Jason
Hi Jason,
Thanks! Long awaited.

For everyone without access to the PDFs:

Major Revelation
The cDNA procedure uses a new "Rapid Library Prep Method" which
(1) Asks for only 500ng of input DNA
(2) Skips the ssDNA step altogether!?!?! Doesn't replace it with suppression PCR either. Pretty much the straight Roe lab methodology there.

No nick-translation, either. Seems like they are counting on that to occur during emPCR?

Anyway, about the cDNA method:

So the issues with cDNA sequence on the 454 were mainly:

(1) polyA tails in high numbers ruin a run
(2) DNA in the 1-2kb range is not efficiently fragmented by nebulization.

The protocol solves the first issue by using random primed 1st strand synthesis. (Downside: will have to get rid of the rRNA prior to 1st strand synthesis. Upside:potentially much less 3' sequence bias.)

Second problem dealt with by fragmenting the RNA with heat+ZnCl2.

Other features:
Starting amount of RNA: 200ng. After fragmentation to mean length of 500 nt that would be about 800 billion molecules. Probably 100 billion are in the desired size range, if all goes well.

No amplification. Minimum acceptable yield of "library" is 7.3 billion molecules. That is a fluorometric determination. So only double stranded molecules will be counted. Of course that includes:

RNA-DNA hybrids. Probably low numbers of these due to the RNaseH activity of AMV Reverse Transcriptase.

dsDNA that has PA on one end and PB on the other.
dsDNA that has PA or PB on both ends
dsDNA with PA or PB on one end and no adaptor on the other
dsDNA with no adaptors

Well, it is supposed to be "Rapid". Interesting...

--
Phillip

Last edited by pmiguel; 12-03-2010 at 05:12 AM. Reason: To disable smilies in text!
pmiguel is offline   Reply With Quote