Ribosome Profiling / Circularization of Library?

[cjang]

Hi,

I am in the process of making a library for the purposes of sequencing ribosome protected fragments, and many of the protocols I have found call for the circularization of the DNA before PCR amplification. Is there a particular reason why you have to circularize?

[pmiguel]

Hi cjang,
You increase your chances of getting a response if you give a little more background than you do above. A link to a protocol you are asking about, at minimum. Like this link.

This is apparently one of those methods to get adapters on both ends of a set of complex polynucleotides, anchored only by a poly-A tail. Never seen this one before, but evidently involves priming 1st strand synthesis with a adaptered oligo-dT primer with a specialized base. A "d-spacer furan".

After 1st strand synthesis, all cDNA will be 3' tailed by this adapter. But to do PCR you need a 5' adapter as well. How to get that? In this case the ssDNA cDNA is circularized -- ligated 5' end to 3' end, and then linearized by a process (using some abasic site nuclease) that cleaves at the "d-spacer furan". Since the "d-spacer furan" is in the middle of the oligo, it has the effect of adding an adapter to both ends of each cDNA.

Actually, adding the abasic site to the adapter seems like prestidigitation to me. But it would have the effect of converting inter-cDNA ligations (unintended side-products) and intra-cDNA ligations into legitimate library molecules. Clever. Maybe too clever, maybe not. Okay, to expand a little -- what would the effect of not doing the linearization step? PCR tends to favor smaller products, so the risk of getting linear concatamers (chimerics) seems low. It would help with adapter-dimers though, probably.

--
Phillip

[pbluescript]

You should contact the authors of the paper. There is a newer version of the protocol out there that has been modified quite a bit from the original Science paper.

[EdK]

Hi cjang,
did you manage to get good quality data from your ribosome profiling seq?
I have ribosome profiling data obtaiend without any circularization step.
I was wondering what kind of algorithm you are using to call your peaks.
Please could you let me know if there is anything freely available already?
Thanks!

[scooter]

Epicentre now has a kit for Ribosome profiling. Here is a link if anyone is interested:

http://www.epibio.com/products/rna-sequencing/ribosome-profiling/artseq-ribosome-profiling-kits

[Pseudoplanet]

Hi all,
did anybody ever use the kit before?
Is there any protocol specific for bacteria?
Many thanks

[tj45]

The circularisation method reduces bias introduced during the ligation step as it only uses Rnl2 for the initial ligation and then Circligase to result in the Sequencing Forward primer site upstream of the fragtment

[Genohub]

reducing bias

T4 RNL2 also introduces a significant amount of ligation bias. To reduce this further, you can use randomized bases at the 5' end of the adapter. Here's a paper that describes this: http://www.ncbi.nlm.nih.gov/pubmed/21890899

- Genohub