Answers.Body: <p>Align all reads from a sample at the same time IMO. This will give you all of the information at once, and it should not be difficult. It should be very obvious whenever there's a disagreement between reads or between reads and reference.</p>
  <p>I would also recommend inspecting chromatograms directly at all sites of interest (such disagreements, insertion sites, possible errors or mutations).</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2021-05-08T01:23:24.613Z
Answers.LastActivityDate: 2021-05-08T01:23:24.613Z
Answers.UserDisplayName: Maximilian Press
Body: <p>I am analyzing Sanger sequencing data to verify whether or not the correct protein coding sequence was inserted during cloning.</p>
  <p>For each sample, I have both forward and reverse Sanger sequencing reads that cover the coding sequence.</p>
  <p>Would it be better if I first align the forward and reverse reads to get a consensus, and then align the consensus to the reference?
  Or should I just align each read directly to the reference?</p>
  
Tags: <phylogenetics>
Title: Alignment to verify plasmid from forward and reverse Sanger sequencing reads
ThreadText: # Alignment to verify plasmid from forward and reverse Sanger sequencing reads
  
  (Asked by: username_0 on 2021-05-07)
  
  I am analyzing Sanger sequencing data to verify whether or not the correct protein coding sequence was inserted during cloning.
  
  For each sample, I have both forward and reverse Sanger sequencing reads that cover the coding sequence.
  
  Would it be better if I first align the forward and reverse reads to get a consensus, and then align the consensus to the reference?
  Or should I just align each read directly to the reference?
  
  **Tags:** phylogenetics
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2021-05-08. Score: 2
  
  Align all reads from a sample at the same time IMO. This will give you all of the information at once, and it should not be difficult. It should be very obvious whenever there's a disagreement between reads or between reads and reference.
  
  I would also recommend inspecting chromatograms directly at all sites of interest (such disagreements, insertion sites, possible errors or mutations).
  
  
  ---
  
  