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

[shahinda rezk]

SOLiD

please i want to know which base shall i know to begin translating the colours to base sequence?

[carmeyeii]

Hi!

I'm analyzing a "second-hand" dataset generated using SOLiD 4. It is a transcriptome mate pair library that is 52 x 37 nt, and I cannot for the sake of me find the protocol that was used to generate those specific read lengths. I have F3 and R3 reads, so I am assuming it is a circularization protocol, but I do not know what the size selection parameters were, or how the circles were cut to produce the final fragments. This info would be very valuable for a more accurate mapping.

Any knowledge would be greatly appreciated!

Thanks a lot,

Carmen

[hoagiang]

which adapter to start sequencing, P1 or P2 primer?

Hi,

I'm using SOLiD SAGE. The SAGE method seems to preserve the strand information. The only unclear question is which primer (or adapter) SOLiD uses to start the sequence. According to the chemistry, it is likely the P1 primer and reading from 3' to 5'. Can anyone confirm it?
I stuck with this for a long time, hopefully someone can have an answer,

Thanks so much,
Hoa

[rivabros]

SOLiD quality data box-plots.... strange?

hi everyone,
I have used the SOLiD2std.pl to change the csfasta and qual files to standard fastq files.
I then ran fastqc to view boxplots of quality data and got these results, attached.
They seem to have poor quality every 5 nt, as if the primer 5 of the procedure failed....
Has anyone seen this type of quality plots before???
I am new with solid data, have worked with ilumina and 454 before.
Thanks for any guidance in advance.
cheers
maximo

[snetmcom]

ligase binding is most efficient at 8 bases. So you need 8mer probe.

[sarde]

Hi,
can you tell me why probes has 8 nucleotides? what´s a function of non-coding bases? thanks

[westerman]

I should point out that the very first dinuc in the columns under 'Template Sequence' in figure 5 is incorrect. It should be 'AT' instead of 'TA'. Looking at the '1st base vs. 2nd base' diagram to the left of 'Template Sequence' shows this.

[westerman]

This newbie will use the color sequence you entered is a good example of why the color scheme depicted in Fig 5 cannot be correct.

RRGBGYY can indicate either the base sequence you show: ATACCATC or the sequence CGCAACTC.

Not at all. The initial base *must* be an 'A' because that is what I defined it as. Once you have the initial base then the rest follows unambiguously. I gave you aRRGBGYY with the small 'a' representing the initial starting base of 'A'. I did not give you an unattached 'RRGBGYY'.

You have the correct underlying idea that color-space can be ambiguous if you start off with no-predefined starting base or if you go 'off-frame'. But when working with color-space you always either (1) start with a pre-defined base or (2) do mapping, in color-space, against a reference thus keeping yourself 'in-frame'.

The above is why everyone says "do work in color-space when you have color-space reads; do not translate into base-space as your initial step." See the many postings on SeqAnswers in regards to this.

[doctortrout]

This newbie will use the color sequence you entered is a good example of why the color scheme depicted in Fig 5 cannot be correct.

RRGBGYY can indicate either the base sequence you show: ATACCATC or the sequence CGCAACTC.

[westerman]

@doctortrout

I believe you have this reversed. If you assigned the same color to all dinucs that started off with the same base then you would be ambiguous. Assigning the same color to different starting bases prevents ambiguous assignments. Of course ideally there there would be 16 colors. But going with 4 colors then as an example let's say we have the sequence (R=>red, G=>green, B=>blue, Y=>yellow):

aRRGBGYY

With 'a' as the very starting base (we need to know this for color-space). We can then read this as bases:

aTACCATC


If 'Red' was assigned to all dinucs starting with an 'A'; e.g., AA, AC, AG, AT then we would read the colors as:

a???????

In other words what second base would the first Red indicate?

Color-space can be very powerful however I find it best for newbies to sit down and write down conversions from color-space to base-space and back in order to grasp the power.

[doctortrout]

Color scheme correction?

I am new to this next gen sequencing, so thanks for providing such a detailed and clear explanation. However, I found Fig. 5 confusing. I believe that a given fluorescent color needs to be assigned to dinucleotides that start with the same base (eg. AN = red) otherwise the sequence will have ambiguities. For example, if the sequence is AG, red would indicate AN. If you want to know N, then all dinucs starting with a G must have the same color (GN = one color, not red).

[navincish]

Noise to signal ratio of SOLiD 4.0

N2S ratio of WFA more than 15% of the sample, is it suitable for sequencing run, however on the axis have very good intensity and in center of axis TXR fluorescence is slight increased.

[analyst]

Is it me or there is an error in the Fig. 5 under template sequence.

for possible dinucleotides for red color should be AT TA CG GC not TA TA CG GC

Since it is an image from ABI's document, the error is actually in the source.

[laxsil]

Hello!
am very new to the abi SOLId technology and am trying to understand things. Could anyone explain wat the bridge probes are?? And also the difference between degenerate bases and universal bases in the dibase probes

[cpnine]

eventually, I find it...