[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

[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..

[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

[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.

[cmebai]

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 ?

[drambald]

May be is a stupid question but...

Hello, I a the following question, regarding the use of AB Solid for ChIP-seq analysis.

To my understanding:

we tag a protein with an antibody, dismantle the cells, denaturate and sonicate the DNA, collect the fragments that were attached to the protein which was attached to the antibody and sequence them.

Problem is: the fragments from sonication should be 100-500 bp long, we only sequence 50 bases at each read: when and how does this further "reduction" take place ?

What should happen is:
1. The fragments are attached to beads
2. Amplification takes place on attached fragments
3. The bead ends up looking like an octopus with N copies of the same fragment
4. Those copies are actually sequenced, but due to technical limitations only the first 50 bases can be read

is this correct?

best regards

[lily Michelle]

Hi all,

Is the flurosence attached to the base or the phosphate group?

thanks

lily

[samanta]

Color space

Hello all,

I wrote this up on color space - nucleotide space conversion and added few Perl scripts to help you proceed.

http://www.homolog.us/blogs/?p=27

Please feel free to comment.

Manoj

[pmiguel]

Hi All,

back to the chemistry of SOLiD. With mate-paired libraries after the 'first mate' comes an internal adaptor. Do I think right that the 'second mate' is sequenced the same way but with primers matching with the internal adaptor?

Anago

Yes, the "R3" mate-pair reads are primed out of the internal adaptor.

--
Phillip

[anago]

Hi All,

back to the chemistry of SOLiD. With mate-paired libraries after the 'first mate' comes an internal adaptor. Do I think right that the 'second mate' is sequenced the same way but with primers matching with the internal adaptor?

Anago

[deepak_bala]

the first base is given to you in your files, but AGAIN, you do not want to decode color space to read your sequence. Align your sequence in color space first.

Thanks for the pointer. I will.

[snetmcom]

Thanks for the reply. I was looking at it and maybe we can determine what the first base is with data from the primer 1 reads from primers n and (n-1).

Correct me if I am wrong, anyone.

the first base is given to you in your files, but AGAIN, you do not want to decode color space to read your sequence. Align your sequence in color space first.

[deepak_bala]

You're right, and you do know the first base based on the adapter/primer you use. I'm sure someone will chime in soon with a more detailed answer....

Thanks for the reply. I was looking at it and maybe we can determine what the first base is with data from the primer 1 reads from primers n and (n-1).

Correct me if I am wrong, anyone.

[ECO]

Hey guys, I need a clarification about the two-base encoding (should be called 'decoding'!). Maybe I haven't understood correctly, but we need to know the first base to be able to decode correctly, right? I am not clear how one would decide/know what base comes first, 'cos I think it is critical to decoding the sequence.

Thanks for any help!

You're right, and you do know the first base based on the adapter/primer you use. I'm sure someone will chime in soon with a more detailed answer....

[deepak_bala]

Hey guys, I need a clarification about the two-base encoding (should be called 'decoding'!). Maybe I haven't understood correctly, but we need to know the first base to be able to decode correctly, right? I am not clear how one would decide/know what base comes first, 'cos I think it is critical to decoding the sequence.

Thanks for any help!