Questions about ABI principle

[sbrohee]

Hi all,

Even if I am quite new to sequencing, my boss and I decided to initiate talks about next-gen sequencing in our university so that we could stay updated.

I am preparing a talk where I am briefly describing the three most known technologies (454, Illumina and SOLiD). While I think I got the principle of the two first approaches, I still have some questions about the SOLiD approach. On the web, I could find explanations going in every directions.

So... I decided it it would be easier to ask experts (hopefully, they have time to answer me).

Of course, if someone knows a very good website / paper explaining very clearly the SOLiD technologies, please give me the URL!

My questions :

a) At each step of the cycle, are the 16 possible oligonucleotides added or only four, then which one?
b) Which bases of the oligonucleotides are the "interesting" one? The first and the second or the fourth and the fifth?

I may have more questions ... but this is already a good start. Many many thanks to you all for your help.

Regards

[pmiguel]

My questions :

a) At each step of the cycle, are the 16 possible oligonucleotides added or only four, then which one?

All 16 are added. But they are labelled systematically with 4 different fluors.
Note that, in principle, this is not necessary. The original protocol, as developed in the Church lab, used single, rather than dual base "encoding". The idea was that additional error detection capability was possible with dual base encoding, even without doing extra cycles.

b) Which bases of the oligonucleotides are the "interesting" one? The first and the second or the fourth and the fifth?

The first and second. In the very early days (v1 chemistry, I think) other bases were used. But the highest accuracy was seen to derive from oligos with the interrogating bases immediately next to the ligation junction.

--
Phillip

[ulz_peter]

You should consider mentioning the Ion Torrent system and perhaps the PacBio systemas well, as they are already commercially available and at least Ion Torrent sells quite some machines (based on rumours at least).

[sbrohee]

@Pmiguel

HI Philip. Many thanks for these clear and fast answers. This is very helpful.

@ulz_peter

Thank you for the advice. I will mention them but without entering into details (not the time to do that).

[mbblack]

Hi all,

Of course, if someone knows a very good website / paper explaining very clearly the SOLiD technologies, please give me the URL!


Regards

I don't have the URL right at hand, but on the ABI web site there is a very detailed white paper explaining the principle of 2 base color coding by Heinz Breu ("A theoretical understanding of 2 base color codes and its application to annotation, error detectiong, and error correction").

P.S. found it - http://www3.appliedbiosystems.com/cm...cms_058265.pdf


Also, Michael Rhodes webinar, third one from the left under the "webinar" tab (http://www.appliedbiosystems.com/abs...-webinars.html) is pretty good at explaining the basics of the chemistry.

[krobison]

Mentioning the Ion Torrent technology in the context of the 454 technology makes it short & easy; the basic principle is the same (flowing unmodified nucleotides) but the detection scheme is different.

PacBio is an easy teaser " simply watch single polymerases add fluorescently-labeled bases in real time" :-)

[newmoon]

Could somebody explain how Solid does a paired-end run? Thanks.
I'm familiar with Illumina.

[pmiguel]

SOLiD actually just anneals a primer to the opposite end of the same ssDNA template and ligates in the opposite direction for their PE reverse read.

Up-side: No need to synthesize a nascent template strand.

Down-side: The reverse direction ligation chemistry does not work as robustly as the forward direction. So the read lengths are shorter. Also, you can't use the new "ECC" ligations if you choose to do PE on the SOLiD.

Note, Mate End (Mate pair) is different. Both reads use forward ligation chemistry so ECC works.

--
Phillip

[newmoon]

Thanks for the explanation