[snetmcom]

Firstly ,thanks for your time of explaining the wonderful tech.And i have a problem ,that is :
At the step of primer reset is it the same strand to be sequenced ?

yes. there are 10,000's of copies of the strand on the bead. That is how the signal is detected.

but to simplify, the original strand is read at reset.

[volks]

4. Those copies are actually sequenced, but due to technical limitations only the first 50 bases can be read

is this correct?

sounds correct to me. have a look at these illustrations, i find them very informative:

http://seq.molbiol.ru/sch_lib_fr.html

http://seq.molbiol.ru/sch_clon_ampl.html

http://seq.molbiol.ru/sch_seq_ligase.html

[KevinLam]

sounds correct to me. have a look at these illustrations, i find them very informative:

http://seq.molbiol.ru/sch_lib_fr.html

http://seq.molbiol.ru/sch_clon_ampl.html

http://seq.molbiol.ru/sch_seq_ligase.html

Nice! Pity the tables look confusing without borders though..

[holywoool]

hi,everyone!

It is said that we are able to detect sequence error when coping with reads in color space var di-base encode methord.And this is true.

But if a sequence error site has a high QUAL value,then can we make a conclusion that this is a sequence error site? If so,does it sound of some controdiction?

What I am confused is that whether we regardless its QUAL value while detecting sequence error ?

Thanks

[cpnine]

eventually, I find it...

[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

[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.

[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.

[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).

[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]

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]

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.

[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.

[sarde]

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

[snetmcom]

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