Tech Summary: ABI's SOLiD (Seq. by Oligo Ligation/Detection), UPDATED for v2.0

[pmiguel]

When converting from colorspace to basespace, if you reach an error (no color, aka "4" or "."), you have 4 possibilities for the rest of the read. How do you determine the correct one?

Example:
Colorspace T000.00
Is basespace TTTTN followed by AA, CC, GG or TT

Which is correct? If you have a sequencing error early in the read, such as position 1 or 2 it goes in the garbage?

Just to reiterate what is said elsewhere in the thread: do not convert out of colorspace prior to alignment! (Instead convert your reference to colorspace.) It is not merely that you lose the benefits of dual base encoding by converting raw reads out of colorspace. Worse, any error in colorspace changes the "color frame--for lack of a better term" for the conversion. This means that any single base sequencing error will propagate through the rest of the read, ensuring that most of the rest of the base space bases are wrong also.

Even if you must use software that is not colorspace-aware, there are tricks you can use to avoid converting out of colorspace.

--
Phillip

[snetmcom]

When converting from colorspace to basespace, if you reach an error (no color, aka "4" or "."), you have 4 possibilities for the rest of the read. How do you determine the correct one?

Example:
Colorspace T000.00
Is basespace TTTTN followed by AA, CC, GG or TT

Which is correct? If you have a sequencing error early in the read, such as position 1 or 2 it goes in the garbage?

You map your reads in colorspace. You do not decode the strand and then map. This gives you the benefits of 2base encoding while detecting errors throughout the tag.

[nilshomer]

When converting from colorspace to basespace, if you reach an error (no color, aka "4" or "."), you have 4 possibilities for the rest of the read. How do you determine the correct one?

Example:
Colorspace T000.00
Is basespace TTTTN followed by AA, CC, GG or TT

Which is correct? If you have a sequencing error early in the read, such as position 1 or 2 it goes in the garbage?

Depends on the alignment tool. Missing colors are not a problem for BFAST since even if there are four possibilities, one typically has a higher likelihood (see the Viterbi algorithm for HMMs). Remember that sequence alignment can be thought of as a path finding problem, or HMM, etc.

[juan]

When converting from colorspace to basespace, if you reach an error (no color, aka "4" or "."), you have 4 possibilities for the rest of the read. How do you determine the correct one?

Example:
Colorspace T000.00
Is basespace TTTTN followed by AA, CC, GG or TT

Which is correct? If you have a sequencing error early in the read, such as position 1 or 2 it goes in the garbage?

[selim27]

Thanks, this information was really helpful! We have been learning about next generation sequencing technologies in class, but our professor's explanations were very vague. I have also read the protocol for illumina/solexa on this forum which was very helpful was well.

[pmiguel]

Perhaps this 4 page article by LifeTech/ABI is what you want. It shows the barcode and mentions that there are two sequencing steps for single runs and three steps for paired runs.

http://www3.appliedbiosystems.com/cm...cms_057554.pdf

Hi Rick,
That is a new feature. Previously there was no bar coding for mate pair runs for the SOLiD--only fragment. But the marketing bulletin you reference above clearly shows a bar-coded mate pair amplicon.

Even more bizarre, the bar code is being read from the P2 primer. This makes no sense within the standard SOLiD amplicon paradigm because P2 is one of the ePCR primers. Nothing can be outside P2 if you amplify with P1 and P2.

Will have to ask our FAS what is up with that.

--
Phillip

[westerman]

Perhaps this 4 page article by LifeTech/ABI is what you want. It shows the barcode and mentions that there are two sequencing steps for single runs and three steps for paired runs.

http://www3.appliedbiosystems.com/cm...cms_057554.pdf

[SOLiD J]

I'm pretty sure it hasn't been mentioned and it's definitely not anywhere on the website but I found out that the barcode is sequenced separately. Turns out there is another universal sequence in between the sequence of interest and the barcode. Another set of universal primers are used to sequence just the barcode. I asked the guy at my campus's nucleic acid facility and he had a manual which illustrated it very well. If anyone finds an online version please post.

[beckham423]

Hello, nice to come here to obtain an access to new sequencing technology

[Svenno]

Hello.
I am studying Biotechnolgy and currently doing a coursework about -Modern Methods of DNA-sequencing-.
Thanks for this helpful explanation, Eco. It realy helped me.
Unfortunately I have problems to understand step 3 in figure 4. Is a Phosphate added to prevent that a Ligation continues?If yes, why? Does it mean that only strangs that start with TA are sequenced in this example. I thought that all 16 combinations have been added.
Or is it just for the case that nothing bound to the bead (seems very unplausible too me)?
Thanks a lot

[rsultana]

Great explanations of the color-space and dibase sequencing.
There is an error though in both Fig.5 of the sticky and Fig.2 of the accompanying SOLiD_Dibase_Sequencing_and_Color_Space_Analysis.pdf - which makes the understanding of the logic difficult.
For the red color, the 4 dinucleotides interrogated by it are listed as "TA CG GC TA" - where the first TA should be AT.
(it just happens that the example in Fig.5/2 starts with AT and it has a red circle associated with it - which makes no sense when you look it in the table above it).
I just thought I might mention this, to make it easier for others to understand the decoding process.

[JackYu]

Thank you very much, Chipper! It's just released. Great!

[Chipper]

Ok, I guess this thread should be updated... Of course anything that can be converted into DNA can be sequenced, I am not sure if they have the kits already but you can read more here:
http://www3.appliedbiosystems.com/AB...ysis/index.htm

[JackYu]

Because I can only see examples for genomic DNA sequencing in the flowchart on the previous page.

[Chipper]

Yes, why not?