Use Analyze -> Align To Reference to align ABI trace files

One of the most common tasks in any molecular biology lab is the need to re-sequence a piece of DNA. Perhaps it is a cloned PCR fragment where you want to confirm the sequence, perhaps you are sequencing across a cloning junction, or maybe screening clones for a successful mutagenesis experiment. Many users immediately think “this is a multiple sequence alignment problem” and so they attempt to align the sequences sent back from the sequencing facility using ClustalW and the Multiple Sequence Alignment interface. However, this suffers from a couple of limitations: (a) ClustalW does not know how to automatically “flip” sequences when you have pairs of reads on opposite strands and (b) the MSA interface doesn’t let you view the raw trace data to help identify sequencing artifacts.

A far better solution is to use the Analyze | Align To Reference function which is tuned for exactly this type of analysis;

  • First open a reference sequence. This might be the PCR fragment you have cloned, or the construct you are re-sequencing.
  • Choose Analyze | Align To Reference.
  • Click on the Add Seqs button and select the ABI (.ab1) files sent by your sequencing facility. You can also use plain sequence files in any format supported by MacVector.
  • Click on the Align button, choose the Sequence Confirmation algorithm and click OK to align all of the reads against the reference.
  • NewImage

    Note that the algorithm automatically “flips” reads to maintain the best alignment and will “clip” out vector sequences (shown in gray text in the image). For more information on the use of the Align To Reference function, please check out the Align To Reference – Sequence Confirmation Tutorial.pdf tutorial that you will find installed in the Applications/MacVector/Documentation/ folder. To understand the different sequence alignment algorithms that are present in MacVector, please check out this blog post.

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