Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
This is a sticky topic.
X
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • pmiguel
    replied
    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.

    --
    Phillip

    Leave a comment:


  • snetmcom
    replied
    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.

    Leave a comment:


  • nilshomer
    replied
    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.

    Leave a comment:


  • juan
    replied
    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?

    Leave a comment:


  • selim27
    replied
    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.

    Leave a comment:


  • pmiguel
    replied
    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.

    --
    Phillip

    Leave a comment:


  • westerman
    replied
    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.

    Thermo Fisher Scientific enables our customers to make the world healthier, cleaner and safer. Delivering technology, pharmaceutical and biotechnology services.

    Leave a comment:


  • SOLiD J
    replied
    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.

    Leave a comment:


  • beckham423
    replied
    Hello, nice to come here to obtain an access to new sequencing technology

    Leave a comment:


  • Svenno
    replied
    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, 12:13 AM.

    Leave a comment:


  • rsultana
    replied
    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.

    Leave a comment:


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

    Leave a comment:


  • Chipper
    replied
    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:
    Thermo Fisher Scientific enables our customers to make the world healthier, cleaner and safer. Delivering technology, pharmaceutical and biotechnology services.

    Leave a comment:


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

    Leave a comment:


  • Chipper
    replied
    Yes, why not?

    Leave a comment:

Latest Articles

Collapse

  • seqadmin
    Techniques and Challenges in Conservation Genomics
    by seqadmin



    The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

    Avian Conservation
    Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
    03-08-2024, 10:41 AM
  • seqadmin
    The Impact of AI in Genomic Medicine
    by seqadmin



    Artificial intelligence (AI) has evolved from a futuristic vision to a mainstream technology, highlighted by the introduction of tools like OpenAI's ChatGPT and Google's Gemini. In recent years, AI has become increasingly integrated into the field of genomics. This integration has enabled new scientific discoveries while simultaneously raising important ethical questions1. Interviews with two researchers at the center of this intersection provide insightful perspectives into...
    02-26-2024, 02:07 PM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 03-14-2024, 06:13 AM
0 responses
34 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-08-2024, 08:03 AM
0 responses
72 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-07-2024, 08:13 AM
0 responses
82 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-06-2024, 09:51 AM
0 responses
68 views
0 likes
Last Post seqadmin  
Working...
X