tRNA sequencing?

[mboth]

Hi,
I was wondering whether anybody has ever wanted to sequence tRNAs? I would be grateful for any information.
I am under the impression cDNA synthesis would be impaired by the modifications and secondary structures of tRNAs, so any tips of how to avoid those pitfalls are greatly appreciated.
I am quite new to this, and I go in right at the deep end!
Thanks
Maike

[krobison]

The PacBio instrument would be particularly interesting to apply this to, since it can read some DNA modifications (indirectly; they cause a characteristic change in the reaction kinetics)

[mrawlins]

On our bacterial samples we see quite a few SOLiD reads that map to tRNA locations. Even when we put the error tolerance to be quite low and the seed size to be high there are a lot more tRNA reads than we would expect due to random matching. It is possible that we are not actually sequencing the tRNA but some pre-tRNA that is not fully processed. Our sample prep is a fairly standard procedure for preparing SOLiD libraries (I don't do the sample prep, but the tech that does has indicated it's using a standard kit from ABI).

While the modifications and secondary structure may impair cDNA synthesis from tRNA, it doesn't seem to be sufficient impairment to completely prevent cDNA synthesis. The intensities may be smaller, but it may still be doable.

That being said, if you have any non-standard bases then I'm not sure how well the sequencers will identify them. They may identify as something else, or they may come back as uncalled bases, depending on the technology used.

[mboth]

Thank you for the comments krobinson and mrawlins! I think we are in a (long) queue for the PacBio machine, and the current strategy is RT at high temperatures. Fingers crossed...

[Zork]

Hi, folks,

We have sequenced tRNAs on a GAII, with mixed results. There seem to be very strong biases in which tRNAs are sequenced; some show up as highly abundant, others extremely rare in the sequenced reads. We know from other methods that these biases don't reflect reality, so *something* is perturbing the library construction or sequencing.

One issue to be aware of is that some tRNAs have the amino acid attached at the 2' carbon, others at the 3' carbon. Not sure about other platforms, but for Illumina RNA sequencing, the adapters are supposed to link to the OH on the 2' carbon (if I remember correctly). Presumably the presence of an amino acid in the way causes trouble with that, and we do see a bias against this class of tRNAs in the sequencing data.

If you're hoping for quantitative data, I'd say you need to consider other methods. If you're just looking for the sequences, you'll probably get great data for some and very little for others. At least, that's what we saw...

Best of luck,

- Gord

[mboth]

Hello Gord,
Thanks for this insight. Would it be possible to pick your brains regarding the protocols for this endeavour? I am doubtful about several steps, eg shearing, the adapter ligation, whether to use stem loop primers or not, and which enzyme to use for the RT? It would help me a great deal to get some advice from someone who's done it before - but I can understand if you are not in a position to share this with me. My email address is [email protected]. Will be glad to hear from you.
Maike