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Old 02-08-2009, 05:00 AM   #21
hongbo
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Default hi

thanks for the information!
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Old 03-04-2009, 02:56 PM   #22
JackYu
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Hi,

I'm new to SOLiD technology. Can it be used to sequence transcriptom?
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Old 03-05-2009, 08:00 AM   #23
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Yes, why not?
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Old 03-05-2009, 09:32 AM   #24
JackYu
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Because I can only see examples for genomic DNA sequencing in the flowchart on the previous page.
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Old 03-05-2009, 10:22 AM   #25
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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
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Old 03-05-2009, 11:12 AM   #26
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Thank you very much, Chipper! It's just released. Great!
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Old 04-04-2009, 01:07 PM   #27
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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.
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Old 06-12-2009, 09:27 AM   #28
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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

Last edited by Svenno; 06-13-2009 at 01:13 AM.
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Old 07-15-2009, 11:23 PM   #29
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Hello, nice to come here to obtain an access to new sequencing technology
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Old 11-14-2009, 05:36 PM   #30
SOLiD J
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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.
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Old 11-16-2009, 09:23 AM   #31
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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
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Old 11-16-2009, 09:39 AM   #32
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Quote:
Originally Posted by westerman View Post
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.

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Old 12-03-2009, 07:53 PM   #33
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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.
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Old 12-04-2009, 05:00 PM   #34
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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?
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Old 12-04-2009, 08:44 PM   #35
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Quote:
Originally Posted by juan View Post
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.
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Old 12-05-2009, 09:00 AM   #36
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Quote:
Originally Posted by juan View Post
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.
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Old 12-07-2009, 04:06 AM   #37
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Quote:
Originally Posted by juan View Post
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.

--
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Old 12-29-2009, 04:09 PM   #38
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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!
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Old 12-29-2009, 06:15 PM   #39
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Quote:
Originally Posted by deepak_bala View Post
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....
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Old 12-29-2009, 06:23 PM   #40
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Quote:
Originally Posted by ECO View Post
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.
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