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

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  • #31
    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?

    Comment


    • #32
      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.

      Comment


      • #33
        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.

        Comment


        • #34
          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

          Comment


          • #35
            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!

            Comment


            • #36
              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....

              Comment


              • #37
                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.

                Comment


                • #38
                  Originally posted by deepak_bala View Post
                  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.

                  Comment


                  • #39
                    Originally posted by snetmcom View Post
                    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.

                    Comment


                    • #40
                      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

                      Comment


                      • #41
                        Originally posted by anago View Post
                        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

                        Comment


                        • #42
                          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
                          http://homolog.us

                          Comment


                          • #43
                            Hi all,

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

                            thanks

                            lily

                            Comment


                            • #44
                              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

                              Comment


                              • #45
                                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 ?

                                Comment

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