text: user16732: I am a student in a Cancer lab. Working with sanger is new to me. While analyzing a report we found an insertion that has not been reported in any databases so far, we were working on checking if the insertion is pathogenic or not but when we reanalyzed the sanger annotation through variant reporter of the insertion it was giving it one position ahead and we analyzed that with the VEP as well that is were the confusion started.
  The sequencer showed annotation of an insertion not between the bases but a step/position next to it:
  c.5532-5533insGAA is the insertion position came after position 32 and before 33 but the sequencer annotates it like c.5533-5534insGAA.
  We are working on human genome hg38.
  Should I consider it a machine error or should I take that position for the result? Or is there a nomenclature error I am making here?
  And
  the annotation is in this form [c.33-34insAUU]+[=] and I don't know what the +[=] represents here.
  
  user3848: Just to give my thoughts:
  
  The missing information is the actual sequence
  
  
  Is this a conflict in the change in position of the insert?
  Could it simply be a consequence of down stream numbering, either a logical error (coding stuff), or lab artefact.
  
  My bet is its point 2, but really we don't know except for the OP because we don't know the sequence.
  Sources of error, Point 2
  
  Sanger sequencing was traditionally known for "frame-shift" errors if there were mono-mers, e.g. AAAA -> AAAAA, it can mess up over the number of bases in a mono-mer.
  PCR amplification can also cause slippage against monomers, same thing as the above point - but it's real.
  1 vs 0 ... this is because in a Pythonic list or any C/Perl-based array numbering starts at 0 (not 1) - hence @terdon's comments.
  
  If upstream mono-mers are present that might be the source of the confusion here and given the amount of sequence data thats very likely.
  What I'd do:
  
  Just Blast the new sequence and see what emerges: if it is genuinely novel - they'll be zero 100% identity hits.
  VCFs ... I think this is VarSome's stuff (forget).
  
  If there are Blast hits it will identify whether there is any upstream lab artefacts.
  Conclusion
  If this was any other situation in human genetics, or even mammalian genetics I'd be excited. However, in this case - I think its a routine result. Firstly, the chance of finding a new "driver" mutation are slim. Secondly, I've looked at this chromosome in a cancer patient (only once) and it was nuts (and thats an under statement) - I've a fairly good memory for numerical patterns, and I'd never seen a pathogenic species behaving like that. I think this insert is just a consequence of the entire mutational chaos that are cancer cells.
  Just to make it clear: I don't do cancer but if you want an authoritative assessment and think you've something major I'd contact (or your PI) the Broad Institute via Geraldine A. Van der Auwera who also has an account on this site via @Geraldine_VdAuwera. Sanger sequencing is not what they do, but they'd know at the drop of a hat what the significance and if they don't respond - thats your answer ;-). Alternatively, VarSome would know, albeit they're commercial enterprise offering freeware.
  
original_text: 16732: <p>I am a student in a Cancer lab. Working with sanger is new to me. While analyzing a report we found an insertion that has not been reported in any databases so far, we were working on checking if the insertion is pathogenic or not but when we reanalyzed the sanger annotation through variant reporter of the insertion it was giving it one position ahead and we analyzed that with the VEP as well that is were the confusion started.</p>
  <p>The sequencer showed annotation of an insertion not between the bases but a step/position next to it:</p>
  <p><strong>c.5532-5533insGAA</strong> is the insertion position came after position 32 and before 33 but the sequencer annotates it like <strong>c.5533-5534insGAA</strong>.</p>
  <p>We are working on human genome hg38.</p>
  <p>Should I consider it a machine error or should I take that position for the result? Or is there a nomenclature error I am making here?</p>
  <p>And
  the annotation is in this form [c.33-34insAUU]+[=] and I don't know what the +[=] represents here.</p>
   3848: <p>Just to give my thoughts:</p>
  <ul>
  <li>The missing information is the actual sequence</li>
  </ul>
  <ol>
  <li>Is this a conflict in the change in position of the insert?</li>
  <li>Could it simply be a consequence of down stream numbering, either a logical error (coding stuff), or lab artefact.</li>
  </ol>
  <p>My bet is its point 2, but really we don't know except for the OP because we don't know the sequence.</p>
  <p><strong>Sources of error, Point 2</strong></p>
  <ul>
  <li>Sanger sequencing was traditionally known for &quot;frame-shift&quot; errors if there were mono-mers, e.g. AAAA -&gt; AAAAA, it can mess up over the number of bases in a mono-mer.</li>
  <li>PCR amplification can also cause slippage against monomers, same thing as the above point - but it's real.</li>
  <li>1 vs 0 ... this is because in a Pythonic list or any C/Perl-based array numbering starts at 0 (not 1) - hence @terdon's comments.</li>
  </ul>
  <p>If upstream mono-mers are present that might be the source of the confusion here and given the amount of sequence data thats very likely.</p>
  <p>What I'd do:</p>
  <ul>
  <li>Just Blast the new sequence and see what emerges: if it is genuinely novel - they'll be zero 100% identity hits.</li>
  <li>VCFs ... I think this is VarSome's stuff (forget).</li>
  </ul>
  <p>If there are Blast hits it will identify whether there is any upstream lab artefacts.</p>
  <p><strong>Conclusion</strong>
  If this was any other situation in human genetics, or even mammalian genetics I'd be excited. However, in this case - I think its a routine result. Firstly, the chance of finding a new &quot;driver&quot; mutation are slim. Secondly, I've looked at this chromosome in a cancer patient (only once) and it was nuts (and thats an under statement) - I've a fairly good memory for numerical patterns, and I'd never seen a pathogenic species behaving like that. I think this insert is just a consequence of the entire mutational chaos that are cancer cells.</p>
  <p>Just to make it clear: I don't do cancer but if you want an authoritative assessment and think you've something major I'd contact (or your PI) the Broad Institute via Geraldine A. Van der Auwera who also has an account on this site via @Geraldine_VdAuwera. Sanger sequencing is not what they do, but they'd know at the drop of a hat what the significance and if they don't respond - thats your answer ;-). Alternatively, VarSome would know, albeit they're commercial enterprise offering freeware.</p>
  