<p>Your core question is:</p>
<blockquote>
<p>(mutations, insertion or deletions) that occurred during the molecular cloning process.</p>
</blockquote>
<p>I asked NCBI directly and their response is:</p>
<blockquote>
<p>For natural genetic variation use 'Variation' feature;</p>
</blockquote>
<blockquote>
<p>For 'artificial' genetic variation (lab-produced by genetic modification) then use 'misc_difference'.</p>
</blockquote>
<p>In summary in all scenarios its singly the 'misc_differences' that are used (see Conclusion).</p>
<p>So in my scenario where I want to screen out targeted mutagenesis its the 'misc_difference', i.e. the second option of the two you present. This is also true in your second scenario because it is changes possibly unintentional, possibly random mutagenesis / or just unintentional errors during cloning. Finally, this is also true for SOME of the 'errors' you are reporting in comments:</p>
<ul>
<li>for the first i, ii and iii that appears to be just correcting stuff - administration, just cross-check with those submitting.</li>
<li>for 'variations' the second i and ii in comments, that falls into the 'misc_difference', because a form of mutagenesis, albeit probably unintentional.</li>
</ul>
<hr />
<p><strong>Conclusion</strong>
The 'variation' feature description isn't clear given the description you reported against NCBI's official Helpdesk response. Its a fair question, its a clear answer, but doesn't explain why the description is confusing.</p>
<hr />
<p>On 'errors' of mis-identification, e.g. correctly identified e.g. via local Blast or Blat, e.g. sequenced the wrong target, I would use an internal marker, e.g. <code>returned to contributor for comment</code>. Pass it back to the contributor and ask for their input and depending what they say simply mark the record in the 'Comments section' e.g. <code>originally defined as XX</code>. The contributor could have made all sorts of different errors, e.g. annotation - copied and pasted the wrong stuff - experimental anything and you need to give them a chance to respond. Uploading these records on an external database, i.e. Genbank, needs a joint agreement with all parties concerned because it is certainly valid information for someone.</p>
<hr />
<p>Note, the official blurb <a href="https://www.ncbi.nlm.nih.gov/genbank/genomesubmit_annotation/#disrupted_genes" rel="nofollow noreferrer">here</a>, doesn't appear informative:</p>
<blockquote>
<p>Disrupted genes and gene fragments</p>
<p>Sometimes a genome will have adjacent or nearby genes that seem to be
only part of a protein. In many cases these indicate a possible
problem with the sequence and/or annotation. A related issue is the
presence of internal stop codons in the conceptual translation of a
CDS that looks like it should be a real CDS. These problems may be due
to a variety of reasons, including <strong>mutations</strong> or sequencing or assembly
artifacts. They can be annotated in a number of ways:</p>
<p>Annotate the gene with /pseudo to indicate that there is a problem
with the gene. Note that this qualifier does NOT mean that the gene is
a pseudogene. (see point 2, below, if it is known that the gene IS a
pseudogene) If multiple gene fragments were present initially, then
add a single gene feature which covers all of the potential coding
regions and add the pseudo qualifier. If known, a note qualifier may
be added indicating why this gene is disrupted, for example:</p>
</blockquote>
<pre><code> 1        200     gene
                         gene    phoA
                         gene_desc     alkaline phosphatase
                         locus_tag     OBB_0001
                         pseudo
                         note    nonfunctional due to frameshift
</code></pre>

CC BY-SA 4.0
2022-03-17T14:40:09.783Z
2022-03-17T22:27:23.693Z
2022-03-17T22:27:23.693Z
M__
<h3>Overview</h3>
<p>To properly address the questions, each possible case must be listed:</p>
<ul>
<li><p><strong>Case 1</strong>: divergence between sequences (sequence in the file vs sequence of the molecule); two cases:</p>
<p><strong>a</strong>- (wrong) sequence in the file vs (correct) sequence in the molecule</p>
<p><strong>b</strong>- (&quot;correct&quot;, theoretical) sequence in the file vs (actual, mutated) sequence in the molecule</p>
</li>
<li><p><strong>Case 2</strong>: divergence between a feature annotation in the file vs the correct (&quot;true&quot; or reference) biological feature; three cases:</p>
<p><strong>a</strong>- wrong location of the feature</p>
<p><strong>b</strong>- wrong feature name</p>
<p><strong>c</strong>- variant sequence of the feature</p>
</li>
</ul>
<h3>Short answer</h3>
<p><strong>Case 1a</strong>: The sequence in the GenBank file must be corrected and the <em>version number</em> of the file must be incremented (see <strong>Q2</strong>).</p>
<p><strong>Case 1b</strong>: A new GenBank file consistent with the actual molecule must be created and the <em>misc_difference</em> feature key must be used to annotate the variations where relevant.</p>
<p><strong>Cases 2a and 2b</strong>: Feature information in the GenBank file must be corrected and the <em>Modification Date</em> of the file must be updated (see <strong>Q2</strong>).</p>
<p><strong>Case 2c</strong>: The <em>misc_difference</em> feature must be used to annotate the variation and the <em>Modification Date</em> of the file must be updated if necessary (see <strong>Q2</strong>).</p>
<h3>Long answer</h3>
<h4>Q1. How to handle the situation when there are divergences between a GenBank file and an actual or theoretical molecule?</h4>
<h5>The <em>misc_difference</em> feature key</h5>
<p>First, let's analyse the situation with the <em>misc_difference</em> feature key.
According to the <a href="https://www.insdc.org/documents/feature_table.html#7.2" rel="nofollow noreferrer">INSDC documentation</a>:</p>
<pre><code>Feature Key           misc_difference


Definition            feature sequence is different from that presented 
                      in the entry and cannot be described by any other 
                      difference key (old_sequence, variation, or modified_base);

Optional qualifiers   /allele=&quot;text&quot;
                      /clone=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot; 
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)
                      /phenotype=&quot;text&quot;
                      /replace=&quot;text&quot; 
                      /standard_name=&quot;text&quot;

Comment               the misc_difference feature key should be used to 
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                      use /replace=&quot;&quot; to annotate deletion, e.g. 
                      misc_difference 412..433
                                      /replace=&quot;&quot;  
</code></pre>
<p>From the Definition above, there is still some ambiguity as to what situation is described. Indeed, &quot;<em>feature sequence is different from that presented in the entry</em>&quot; can describe both of the following cases:</p>
<ul>
<li><p>the sequence in the file and/or in the molecule is not the one expected for the feature annotated</p>
</li>
<li><p>the sequence in the molecule is not the one represented in the file</p>
</li>
</ul>
<p><em>Note: In fact, as we will see below, there should be no divergence between the sequence in the file and the sequence of the molecule.</em></p>
<p>As for the Comment that says that &quot;<em>the misc_difference feature key should be used to describe variability that arises as a result of genetic manipulation (e.g. site directed mutagenesis)</em>&quot;, it does not really clarify which of the two cases above is the one intended. Further, the example given in the Comment, which at first sight seems more enlightening, still carries some ambiguity. It says that to annotate a deletion, one should use:</p>
<pre><code>misc_difference 412..433
                /replace=&quot;&quot;
</code></pre>
<p>All in all, it implies that the deletion is in the molecule (vs in the represented sequence), that it arose as a result of genetic manipulation and is therefore indicated in the file as explained above.</p>
<p>However, it is very counter-intuitive, and not very practical when searching sequences of variants among sequence files, to have a sequence file that is supposed to describe a molecule and in which the sequence is actually different from the sequence encountered in the molecule it is supposed to describe.</p>
<p>And indeed, this view goes against common observed recent practice where the <em>misc_difference</em> feature key is used to indicate how to go from the sequence represented in the sequence file to another version:</p>
<ul>
<li><p><strong>Example 1</strong>: <a href="https://www.ncbi.nlm.nih.gov/nuccore/D90147.1" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/D90147.1</a></p>
<pre><code>     misc_difference 308..310
                     /gene=&quot;mucA&quot;
                     /note=&quot;conflict: deletion&quot;
                     /citation=[1]
                     /replace=&quot;&quot;
</code></pre>
<p>The indicated deletion is not in the sequence but is a way to go from the sequence to a previously reported CDS sequence for the annotated gene.</p>
<p>Translation of the CDS sequence in the GenBank file:</p>
<pre><code>MKVDIFESSGASRVHSIPFYLQRISAGFPSPAQGYEKQELNLHEYCVRHPSATYFLRVSGSSMEDGRIHDGDVLVVDRSLTASHGSIVVACIHNEFTVKRLLLRPRPCLMPMNKDFPVYYIDPDNESVEIWGVVTHSLIEHPVCLR*
</code></pre>
<p>Previously reported CDS (see: <a href="https://www.ncbi.nlm.nih.gov/nuccore/M13388;" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/M13388;</a>
hyphen/dash in the sequence added by me to mark the difference)</p>
<pre><code>MKVDIFESSGAS-VHSIPFYLQRISAGFPSPAQGYEKQELNLHEYCVRHPSATYFLRVSGSSMEDGRIHDGDVLVVDRSLTASHGSIVVACIHNEFTVKRLLLRPRPCLMPMNKDFPVYYIDPDNESVEIWGVVTHSLIEHPVCLR*
</code></pre>
<p>Ref.: <a href="https://pubmed.ncbi.nlm.nih.gov/1902211/" rel="nofollow noreferrer">https://pubmed.ncbi.nlm.nih.gov/1902211/</a></p>
</li>
<li><p><strong>Example 2</strong></p>
<p>In each of the GenBank files below, the <em>misc_difference</em> feature key is used to indicate <em>how to go from the (mutated) sequence in the GenBank file to the sequence of the &quot;wild type&quot;</em>.</p>
<ul>
<li><p><em>Escherichia coli</em> <em>uidA</em> gene for beta-glucuronidase mutant E413G, clone M2</p>
<p><a href="https://www.ncbi.nlm.nih.gov/nuccore/AM180563.1" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/AM180563.1</a></p>
<pre><code> misc_difference 1238
               /gene=&quot;uidA&quot;
               /experiment=&quot;experimental evidence, no additional details
               recorded&quot;
               /note=&quot;site directed mutagenesis&quot;
               /compare=S69414.1
               /allele=&quot;wild type&quot;
               /phenotype=&quot;Active GUS&quot;
               /replace=&quot;a&quot;
</code></pre>
</li>
<li><p><em>Escherichia coli</em> <em>uidA</em> gene for beta-glucuronidase mutant C464R, clone M4</p>
<p><a href="https://www.ncbi.nlm.nih.gov/nuccore/AM180564.1" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/AM180564.1</a></p>
<pre><code>misc_difference 1390
                /gene=&quot;uidA&quot;
                /experiment=&quot;experimental evidence, no additional details
                recorded&quot;
                /note=&quot;site directed mutagenesis&quot;
                /compare=S69414.1
                /allele=&quot;wild type&quot;
                /phenotype=&quot;Active GUS&quot;
                /replace=&quot;t&quot;
</code></pre>
</li>
</ul>
<p>See also:</p>
<ul>
<li><p><em>Escherichia coli</em> <em>uidA</em> gene for beta-glucuronidase mutant Y468C, clone M6</p>
<p><a href="https://www.ncbi.nlm.nih.gov/nuccore/AM180565.1" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/AM180565.1</a></p>
</li>
<li><p><em>Escherichia coli</em> <em>uidA</em> gene for beta-glucuronidase mutant W549R, clone M12</p>
<p><a href="https://www.ncbi.nlm.nih.gov/nuccore/AM180566.1" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/AM180566.1</a></p>
</li>
</ul>
</li>
<li><p><strong>Example 3</strong>: <a href="https://www.ncbi.nlm.nih.gov/nuccore/KC999035.4" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/KC999035.4</a></p>
<pre><code> misc_difference 137414..137415

                 /note=&quot;TT compared with single T in original ISCfr1
                 sequence that would result in two partial transposase
                 proteins. Confirmed as correct in raw sequence data.&quot;
</code></pre>
<p>Here again the <em>misc_difference</em> feature key is used to describe <em>how to pass from the represented sequence to a previous sequence version of the considered gene</em>.</p>
</li>
</ul>
<p>So the <em>misc_difference</em> feature key is appropriate when the sequence in the sequence file <em>does</em> match the sequence in the molecule but does <em><strong>not</strong> (any more)</em> match <em>another reference sequence</em> (possibly referred to by another gene-related feature) because variations in the sequence have occurred due to some genetic manipulation or, as in Example 3, due to discrepancy with previous sequencing results.</p>
<p>Thus, the <em>misc_difference</em> feature key is appropriate in <strong>Cases 1b and 2c</strong>, and the <em>/replace</em> qualifier should be used to indicate how to get from the represented sequence to the other reference sequence for the designated feature (in Example 3 above, the <em>/note</em> qualifier is used instead to describe the difference).</p>
<h5>Other cases</h5>
<p>There are three other cases left:</p>
<ul>
<li><strong>Case 1a</strong>: the features in the GenBank file are consistent with the molecule, but the sequence in the file is wrong</li>
<li><strong>Cases 2a and 2b</strong>: the sequence in the GenBank file is consistent with the molecule, but information about some features is wrong</li>
</ul>
<p>Those three cases can all be classified as a GenBank file containing errors (in contrast with <strong>case 1b</strong> where the &quot;error&quot; is in the obtained molecule and with <strong>case 2c</strong> where the information in the GenBank file is merely incomplete). A GenBank file containing errors is just not appropriate, and the solution here is simply to correct the GenBank file to accurately describe the corresponding molecule.</p>
<p>All that remains is to answer to the second question about how to track changes...</p>
<h4>Q2. How to keep track of changes between versions of a GenBank file when corrections or updates are made?</h4>
<h5>Versioning and time-stamping GenBank files</h5>
<p>GenBank files have two fields that can be used to track their history: the <strong>Modification Date</strong> field and the <strong>VERSION</strong> field.</p>
<p>The <strong>VERSION</strong> field uses an <em>accession.version</em> format, where the <em>accession number</em> is fixed and the <em>version number</em> is incremented only when the sequence data is modified.</p>
<p>See:</p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/samplerecord/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/samplerecord/</a></li>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#ModificationDateB" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#ModificationDateB</a></li>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#VersionB" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#VersionB</a></li>
</ul>
<p><em>Note that only modifications in the sequence result in a change in the version number. Changes in annotations only affect the timestamp (Modification Date) of the file.</em></p>
<p>See:</p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/sequenceids/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/sequenceids/</a></li>
</ul>
<p>With this information we can now describe how to properly keep track of the successive versions of a GenBank file in each of the cases described in <strong>Q1</strong>.</p>
<h5>Proper way to keep track of modifications in GenBank files</h5>
<p><strong>Case 1a</strong>: (wrong) sequence in the file vs (correct) sequence in the molecule</p>
<p>The sequence in the file must be corrected and the version number of the file must be incremented by one.</p>
<p>Moreover the respective file versions (the former and the newer) must refer to each other in the COMMENT field of their respective header.</p>
<p>See:</p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/release/current/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/release/current/</a> --&gt; 3.4.2  Entry Organization</li>
</ul>
<p><strong>Case 1b</strong>: (&quot;correct&quot;, theoretical) sequence in the file vs (actual, mutated) sequence in the molecule</p>
<p>A new GenBank file and new molecule name must be created to accurately describe the variant molecule. The <em>misc_difference</em> feature key along with the <em>/replace</em> qualifier must be used as described above where it is deemed appropriate.</p>
<p>The gene-related features affected should keep the same <em>/gene</em> qualifier, but an <em>/allele</em> qualifier might be used to indicate the presence of a difference and the <em>/note</em> and <em>/phenotype</em> qualifiers can be used to indicate the reference sequence to which the allele should be compared to and any possible phenotypic consequence of the variation.</p>
<p>The <em>misc_difference</em> feature key can be given the same gene-related qualifier information as the gene where the difference occurs (<em>/gene</em>, <em>/allele</em>, <em>/note</em>...)</p>
<p>In the special case of variants unintentionally obtained during genetic construction or genetic manipulation, an altered name for the molecule and GenBank file might be used to reflect the fact that the corresponding sequence is different from the theoretically expected sequence, and the <em>misc_difference</em> feature key should be used to indicate any difference that occurred during the construction compared to the initially targetted sequence.</p>
<p><strong>Cases 2a, 2b or 2c</strong>: wrong or incomplete feature data</p>
<p>The feature annotation in the file must be corrected and the <em>Modification Date</em> field must be updated according to the correction date.</p>
<h3>Additional references</h3>
<p><strong>Sequence revision history</strong></p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/genbank/sequencerevisionhistory/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/genbank/sequencerevisionhistory/</a></li>
</ul>
<p><strong>Examples of revision history of some GenBank files</strong></p>
<ul>
<li><a href="https://www.ncbi.nlm.nih.gov/nuccore/U46667?report=girevhist" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/U46667?report=girevhist</a></li>
<li><a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_029716?report=girevhist" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/NM_029716?report=girevhist</a></li>
<li><a href="https://www.ncbi.nlm.nih.gov/nuccore/NM_033116?report=girevhist" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/nuccore/NM_033116?report=girevhist</a></li>
</ul>
<h3>Added for convenience</h3>
<p>Here below is reproduced the description and usage of some <em>feature keys</em> and <em>qualifiers</em> that might be relevant to, or linked with, the question of <em>how to describe and annotate sequence variants</em>.</p>
<p>I grouped those definitions there as it might help one to distinguish the different types of variations and divergences and to understand and chose the appropriate term to annotate sequence variants.</p>
<h4>Selected feature keys</h4>
<p><strong>gene</strong></p>
<pre><code>Feature Key           gene


Definition            region of biological interest identified as a gene 
                      and for which a name has been assigned;

Optional qualifiers   /allele=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /function=&quot;text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot;
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)
                      /operon=&quot;text&quot;
                      /product=&quot;text&quot;
                      /pseudo
                      /pseudogene=&quot;TYPE&quot;
                      /phenotype=&quot;text&quot;
                      /standard_name=&quot;text&quot;
                      /trans_splicing


Comment               the gene feature describes the interval of DNA that 
                      corresponds to a genetic trait or phenotype; the feature is,
                      by definition, not strictly bound to it's positions at the 
                      ends;  it is meant to represent a region where the gene is 
                      located.
</code></pre>
<p><strong>misc_difference</strong></p>
<pre><code>Feature Key           misc_difference


Definition            feature sequence is different from that presented 
                      in the entry and cannot be described by any other 
                      difference key (old_sequence, variation, or modified_base);

Optional qualifiers   /allele=&quot;text&quot;
                      /clone=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot; 
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)
                      /phenotype=&quot;text&quot;
                      /replace=&quot;text&quot; 
                      /standard_name=&quot;text&quot;

Comment               the misc_difference feature key should be used to 
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                      use /replace=&quot;&quot; to annotate deletion, e.g. 
                      misc_difference 412..433
                                      /replace=&quot;&quot;  
</code></pre>
<p><strong>modified_base</strong></p>
<pre><code>Feature Key           modified_base


Definition            the indicated nucleotide is a modified nucleotide and
                      should be substituted for by the indicated molecule
                      (given in the mod_base qualifier value)
 
Mandatory qualifiers  /mod_base=&lt;modified_base&gt;

Optional qualifiers   /allele=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /frequency=&quot;text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot;
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)

Comment               value is limited to the restricted vocabulary for 
                      modified base abbreviations;
</code></pre>
<p><strong>old_sequence</strong></p>
<pre><code>Feature Key           old_sequence


Definition            the presented sequence revises a previous version of the
                      sequence at this location;

Mandatory qualifiers  /citation=[number]
                      Or
                      /compare=[accession-number.sequence-version]

Optional qualifiers   /allele=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot;
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)
                      /replace=&quot;text&quot;

Comment               /replace=&quot;&quot; is used to annotate deletion, e.g. 
                      old_sequence 12..15
                      /replace=&quot;&quot; 
                      NOTE: This feature key is not valid in entries/records
                      created from 15-Oct-2007.
</code></pre>
<p><strong>variation</strong></p>
<pre><code>Feature Key           variation

Definition            a related strain contains stable mutations from the same
                      gene (e.g., RFLPs, polymorphisms, etc.) which differ
                      from the presented sequence at this location (and
                      possibly others);

Optional qualifiers   /allele=&quot;text&quot;
                      /db_xref=&quot;&lt;database&gt;:&lt;identifier&gt;&quot;
                      /experiment=&quot;[CATEGORY:]text&quot;
                      /frequency=&quot;text&quot;
                      /gene=&quot;text&quot;
                      /gene_synonym=&quot;text&quot;
                      /inference=&quot;[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]&quot;
                      /locus_tag=&quot;text&quot; (single token)
                      /map=&quot;text&quot;
                      /note=&quot;text&quot;
                      /old_locus_tag=&quot;text&quot; (single token)
                      /phenotype=&quot;text&quot;
                      /product=&quot;text&quot;
                      /replace=&quot;text&quot;
                      /standard_name=&quot;text&quot;

Comment               used to describe alleles, RFLP's,and other naturally
                      occurring mutations and  polymorphisms; variability arising
                      as a result of genetic manipulation (e.g. site directed
                      mutagenesis) should be described with the misc_difference
                      feature; use /replace=&quot;&quot; to annotate deletion, e.g. 
                      variation   4..5
                                  /replace=&quot;&quot;  
</code></pre>
<h4>Selected qualifiers</h4>
<p><strong>/allele</strong></p>
<pre><code>  Qualifier       /allele=
  Definition      name of the allele for the given gene 
  Value format    &quot;text&quot;
  Example         /allele=&quot;adh1-1&quot;
  Comment         all gene-related features (exon, CDS etc) for a given 
                  gene should share the same /allele qualifier value; 
                  the /allele qualifier value must, by definition, be 
                  different from the /gene qualifier value; when used with 
                  the variation feature key, the allele qualifier value 
                  should be that of the variant.
</code></pre>
<p><strong>/clone</strong></p>
<pre><code>  Qualifier       /clone=
  Definition      clone from which the sequence was obtained
  Value format    &quot;text&quot;
  Example         /clone=&quot;lambda-hIL7.3&quot;
  Comment         not more than one clone should be specified for a given source 
                  feature;  to indicate that the sequence was obtained from
                  multiple clones, multiple source features should be given.
</code></pre>
<p><strong>/compare</strong></p>
<pre><code>  Qualifier       /compare=
  Definition      Reference details of an existing public INSD entry 
                  to which a comparison is made
  Value format    [accession-number.sequence-version]
  Example         /compare=AJ634337.1
  Comment         not to be used for new entries from 14-MAY-2021; This qualifier
                  may be used on the following features: misc_difference, unsure,
                  old_sequence and variation.
                  The feature &quot;old_sequence&quot; must have either a
                  /citation or a /compare qualifier. Multiple /compare
                  qualifiers with different contents are allowed within a 
                  single feature. 
                  This qualifier is not intended for large-scale annotation 
                  of variations, such as SNPs.
</code></pre>
<p><strong>/function</strong></p>
<pre><code>  Qualifier       /function=
  Definition      function attributed to a sequence
  Value format    &quot;text&quot;
  Example         function=&quot;essential for recognition of cofactor&quot;
  Comment         /function is used when the gene name and/or product name do not 
                  convey the function attributable to a sequence.
</code></pre>
<p><strong>/gene</strong></p>
<pre><code>  Qualifier       /gene=
  Definition      symbol of the gene corresponding to a sequence region
  Value format    &quot;text&quot;
  Example         /gene=&quot;ilvE&quot; 
</code></pre>
<p><strong>/locus_tag</strong></p>
<pre><code>  Qualifier       /locus_tag=
  Definition      a submitter-supplied, systematic, stable identifier for a gene
                  and its associated features, used for tracking purposes
  Value Format    &quot;text&quot;(single token) 
                  but not &quot;&lt;1-5 letters&gt;&lt;5-9 digit integer&gt;[.&lt;integer&gt;]&quot;
  Example         /locus_tag=&quot;ABC_0022&quot; 
                  /locus_tag=&quot;A1C_00001&quot;
  Comment         /locus_tag can be used with any feature that /gene can be used
                  with; identical /locus_tag values may be used within an
                  entry/record, but only if the identical /locus_tag values are 
                  associated with the same gene; in all other circumstances the
                  /locus_tag value must be unique within that entry/record.
                  Multiple /locus_tag values are not allowed within one feature for
                  entries created after 15-OCT-2004. 
                  If a /locus_tag needs to be re-assigned the /old_locus_tag
                  qualifier should be used to store the old value. The /locus_tag
                  value should not be in a format which resembles INSD accession
                  numbers, accession.version, or /protein_id identifiers.
</code></pre>
<p><strong>/map</strong></p>
<pre><code>  Qualifier       /map=
  Definition      genomic map position of feature
  Value format    &quot;text&quot;
  Example         /map=&quot;8q12-q13&quot;
</code></pre>
<p><strong>/note</strong></p>
<pre><code>  Qualifier       /note=
  Definition      any comment or additional information
  Value format    &quot;text&quot;
  Example         /note=&quot;This qualifier is equivalent to a comment.&quot;
</code></pre>
<p><strong>/operon</strong></p>
<pre><code>  Qualifier       /operon=
  Definition      name of the group of contiguous genes transcribed into a 
                  single transcript to which that feature belongs.
  Value format    &quot;text&quot;
  Example         /operon=&quot;lac&quot;
</code></pre>
<p><strong>/phenotype</strong></p>
<pre><code>  Qualifier       /phenotype=
  Definition      phenotype conferred by the feature, where phenotype is defined as
                  a physical, biochemical or behavioural characteristic or set of 
                  characteristics
  Value format    &quot;text&quot;
  Example         /phenotype=&quot;erythromycin resistance&quot;
</code></pre>
<p><strong>/product</strong></p>
<pre><code>  Qualifier       /product=
  Definition      name of the product associated with the feature, e.g. the mRNA of
                  an mRNA feature, the polypeptide of a CDS, the mature peptide of
                  a mat_peptide, etc.
  Value format    &quot;text&quot;
  Example         /product=&quot;trypsinogen&quot; (when qualifier appears in CDS feature)
                  /product=&quot;trypsin&quot; (when qualifier appears in mat_peptide
                  feature)
                  /product=&quot;XYZ neural-specific transcript&quot; (when qualifier appears
                  in mRNA feature)
</code></pre>
<p><strong>/pseudo</strong></p>
<pre><code>  Qualifier       /pseudo
  Definition      indicates that this feature is a non-functional version of the
                  element named by the feature key
  Value format    none
  Example         /pseudo
  Comment         The qualifier /pseudo should be used to describe non-functional 
                  genes that are not formally described as pseudogenes, e.g. CDS 
                  has no translation due to other reasons than pseudogenisation
                  events.
                  Other reasons may include sequencing or assembly errors.
                  In order to annotate pseudogenes the qualifier /pseudogene= must
                  be used indicating the TYPE which can be taken from the INSDC
                  controlled vocabulary for pseudogenes.
</code></pre>
<p><strong>/pseudogene</strong></p>
<pre><code>  Qualifier       /pseudogene=
  Definition      indicates that this feature is a pseudogene of the element
                  named by the feature key
  Value format    &quot;TYPE&quot;
                  where TYPE is one of the following:
                  processed, unprocessed, unitary, allelic, unknown

  Example         /pseudogene=&quot;processed&quot;
                  /pseudogene=&quot;unprocessed&quot;
                  /pseudogene=&quot;unitary&quot;
                  /pseudogene=&quot;allelic&quot;
                  /pseudogene=&quot;unknown&quot;

  Comment         TYPE is a term taken from the INSDC controlled vocabulary for
                  pseudogenes (http://www.insdc.org/documents/pseudogene-
                  qualifier-vocabulary):

                  processed: the pseudogene has arisen by reverse transcription
                  of a mRNA into cDNA, followed by reintegration into the genome.
                  Therefore, it has lost any intron/exon structure, and it might
                  have a pseudo-polyA-tail.

                  unprocessed: the pseudogene has arisen from a copy of the
                  parent gene by duplication followed by accumulation of random
                  mutations.
                  The changes, compared to their functional homolog, include
                  insertions, deletions, premature stop codons, frameshifts and a
                  higher proportion of non-synonymous versus synonymous
                  substitutions.
                  
                  unitary: the pseudogene has no parent. It is the original gene,
                  which is functional is some species but disrupted in some way
                  (indels, mutation, recombination) in another species or strain.

                  allelic: a (unitary) pseudogene that is stable in the
                  population but importantly it has a functional alternative
                  allele also in the population. i.e., one strain may have the
                  gene, another strain may have the pseudogene.
                  MHC haplotypes have allelic pseudogenes.

                  unknown: the submitter does not know the method of
                  pseudogenisation.
</code></pre>
<p><strong>/standard_name</strong></p>
<pre><code>  Qualifier       /standard_name=
  Definition      accepted standard name for this feature
  Value format    &quot;text&quot;
  Example         /standard_name=&quot;dotted&quot;
  Comment         use /standard_name to give full gene name, but use /gene to
                  give gene symbol (in the above example /gene=&quot;Dt&quot;).
</code></pre>

CC BY-SA 4.0
2022-04-24T18:26:09.317Z
2022-05-02T08:58:27.963Z
2022-05-02T08:58:27.963Z
The Quark
<h3>EDIT</h3>
<p>By thinking over the issue, I realized it was mixing two linked but separate questions:</p>
<ul>
<li><p><strong>Q1. How to handle the situation when there are divergences between a GenBank file and an actual or theoretical molecule:</strong></p>
<ul>
<li><p>Should the difference be indicated in the feature table? If so, with which feature key? And if not, how should it be indicated or what should be done?</p>
</li>
<li><p>Further, when indicating a divergence using a feature key, which should be considered the reference: the file or the molecule?</p>
</li>
</ul>
</li>
<li><p><strong>Q2. How to keep track of changes between versions of a GenBank file when corrections or updates are made?</strong></p>
</li>
</ul>
<p>I have thus re-titled the question accordingly, and by making further researches into the issues I found what seems to me the appropriate solutions (see <a href="https://bioinformatics.stackexchange.com/a/18978/15221">my answer</a> below).</p>
<h3>ORIGINAL QUESTION</h3>
<p><strong>Background</strong> I am maintaining a database of sequence files describing a collection of bacterial strains and plasmids that we have in our lab.</p>
<p>I am confronted to two sorts of issues:</p>
<ul>
<li>some sequence files contain errors (see comments below for examples of errors)</li>
<li>other sequence files describe theoretical sequences of molecules that themselves contain variations (mutations, insertion or deletions) that occurred during the molecular cloning process.</li>
</ul>
<p><strong>Objective</strong> I want to annotate the sequences to mark those errors and variations using the GenBank format. However, from the feature keys of the GenBank format described on the <a href="https://www.insdc.org/documents/feature_table.html" rel="nofollow noreferrer">INSDC website</a>, it is not clear to me which feature key should be used in each case, essentially this is either 'variation' or 'misc_description'.</p>
<p><strong>Example</strong> To better explain the confusion, here below are what seems to me the most relevant feature keys, and my comment of the problem I have with them:</p>
<pre><code>Feature key           variation
Definition            a related strain contains stable mutations from the same
                      gene (e.g., RFLPs, polymorphisms, etc.) which differ
                      from the presented sequence at this location (and
                      possibly others);
...
Comment               used to describe alleles, RFLP's,and other naturally 
                      occurring mutations and  polymorphisms;
                  &gt;&gt;&gt; variability arising as a result of genetic 
                      manipulation (e.g. site directed mutagenesis) should 
                      be described with the misc_difference feature;
</code></pre>
<p>According to the line marked <code>&gt;&gt;&gt;</code> above, the feature key <code>misc_difference</code> should be used...</p>
<pre><code>Feature Key           misc_difference
Definition            feature sequence is different from that presented in the
                      entry and cannot be described by any other difference
                      key (old_sequence, variation, or modified_base);
...
Comment               the misc_difference feature key should be used to
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                  &gt;&gt;&gt; use /replace=&quot;&quot; to annotate deletion, e.g.
                      misc_difference 412..433
                                      /replace=&quot;&quot;  
</code></pre>
<p>But now, according to the example marked <code>&gt;&gt;&gt;</code> above, it means the variation (here a deletion) is NOT in the considered entry...</p>
<p><strong>Question</strong> So how are variations (mutation, insertion, deletion, errors) correctly described in Genbank given that are actually present in an entry ?</p>

CC BY-SA 4.0
2022-03-17T13:32:48.800Z
This is a good question. One thing I'm puzzled by are 'some sequence files contain errors' if its an ambiguity that is simply dealt with using the degenerate code, e.g. R for A or G ambiguity, N for total ambiguity. If its formatting errors within the file, I'd personally remove them.
M__
CC BY-SA 4.0
2022-03-17T13:49:49.230Z
@M__ Thank you for the comment. By "errors" I don't mean "ambiguities", I mean sequence files that are not accurate in the sense that the sequence they contain is different from the sequence carried by the molecule they are supposed to describe as checked by sequencing. One could then just correct or discard the files, but for some of them, for the sake of archiving and keeping track of the developments, I would like to be able to annotate those errors.
The Quark
CC BY-SA 4.0
2022-03-17T15:24:26.203Z
@M__ Following up your (temporary ?) answer below: * Examples of errors: (i) a feature is properly annotated, but the sequence given is incorrect, (ii) a feature is correctly mentioned, but the sequence span is incorrect (iii) the name of a feature is incorrect (wrong variant for example). * Examples of "variations": (i) during the cloning process a T mutated to a G (variation = 'mutation') (ii) during the cloning process some bases got inserted/deleted (variation = 'insertion' or 'deletion' or 'indel'). Let me know if more details can help.
The Quark
CC BY-SA 4.0
2022-03-17T19:00:20.240Z
@M__ That's true, I can see that... Yes, I think too that the most important aspects are mutations (variations), seconded by errors in the sequence data (over annotation errors). Thank you very much again for looking into this.
The Quark
CC BY-SA 4.0
2022-03-17T21:20:42.203Z
okay post-updated, official response below.
M__
<file-formats><genbank>
How to properly annotate sequence variants and errors in a GenBank file format and how to keep track of successive versions of a GenBank file
# How to properly annotate sequence variants and errors in a GenBank file format and how to keep track of successive versions of a GenBank file

(Asked by: username_0 on 2022-03-17)

EDIT
By thinking over the issue, I realized it was mixing two linked but separate questions:

Q1. How to handle the situation when there are divergences between a GenBank file and an actual or theoretical molecule:

Should the difference be indicated in the feature table? If so, with which feature key? And if not, how should it be indicated or what should be done?

Further, when indicating a divergence using a feature key, which should be considered the reference: the file or the molecule?



Q2. How to keep track of changes between versions of a GenBank file when corrections or updates are made?


I have thus re-titled the question accordingly, and by making further researches into the issues I found what seems to me the appropriate solutions (see my answer below).
ORIGINAL QUESTION
Background I am maintaining a database of sequence files describing a collection of bacterial strains and plasmids that we have in our lab.
I am confronted to two sorts of issues:

some sequence files contain errors (see comments below for examples of errors)
other sequence files describe theoretical sequences of molecules that themselves contain variations (mutations, insertion or deletions) that occurred during the molecular cloning process.

Objective I want to annotate the sequences to mark those errors and variations using the GenBank format. However, from the feature keys of the GenBank format described on the INSDC website, it is not clear to me which feature key should be used in each case, essentially this is either 'variation' or 'misc_description'.
Example To better explain the confusion, here below are what seems to me the most relevant feature keys, and my comment of the problem I have with them:
Feature key           variation
Definition            a related strain contains stable mutations from the same
                      gene (e.g., RFLPs, polymorphisms, etc.) which differ
                      from the presented sequence at this location (and
                      possibly others);
...
Comment               used to describe alleles, RFLP's,and other naturally 
                      occurring mutations and  polymorphisms;
                  >>> variability arising as a result of genetic 
                      manipulation (e.g. site directed mutagenesis) should 
                      be described with the misc_difference feature;

According to the line marked >>> above, the feature key misc_difference should be used...
Feature Key           misc_difference
Definition            feature sequence is different from that presented in the
                      entry and cannot be described by any other difference
                      key (old_sequence, variation, or modified_base);
...
Comment               the misc_difference feature key should be used to
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                  >>> use /replace="" to annotate deletion, e.g.
                      misc_difference 412..433
                                      /replace=""  

But now, according to the example marked >>> above, it means the variation (here a deletion) is NOT in the considered entry...
Question So how are variations (mutation, insertion, deletion, errors) correctly described in Genbank given that are actually present in an entry ?

**Tags:** file-formats, genbank
> **Comments:**
> **username_1**: This is a good question. One thing I'm puzzled by are 'some sequence files contain errors' if its an ambiguity that is simply dealt with using the degenerate code, e.g. R for A or G ambiguity, N for total ambiguity. If its formatting errors within the file, I'd personally remove them.  
> **username_0**: @M__ Thank you for the comment. By "errors" I don't mean "ambiguities", I mean sequence files that are not accurate in the sense that the sequence they contain is different from the sequence carried by the molecule they are supposed to describe as checked by sequencing. One could then just correct or discard the files, but for some of them, for the sake of archiving and keeping track of the developments, I would like to be able to annotate those errors.  
> **username_0**: @M__ Following up your (temporary ?) answer below: * Examples of errors: (i) a feature is properly annotated, but the sequence given is incorrect, (ii) a feature is correctly mentioned, but the sequence span is incorrect (iii) the name of a feature is incorrect (wrong variant for example). * Examples of "variations": (i) during the cloning process a T mutated to a G (variation = 'mutation') (ii) during the cloning process some bases got inserted/deleted (variation = 'insertion' or 'deletion' or 'indel'). Let me know if more details can help.  
> **username_0**: @M__ That's true, I can see that... Yes, I think too that the most important aspects are mutations (variations), seconded by errors in the sequence data (over annotation errors). Thank you very much again for looking into this.  
> **username_1**: okay post-updated, official response below.  

---

## 2 answers

### Answer by username_1 on 2022-03-17. Score: 1

Your core question is:

(mutations, insertion or deletions) that occurred during the molecular cloning process.

I asked NCBI directly and their response is:

For natural genetic variation use 'Variation' feature;


For 'artificial' genetic variation (lab-produced by genetic modification) then use 'misc_difference'.

In summary in all scenarios its singly the 'misc_differences' that are used (see Conclusion).
So in my scenario where I want to screen out targeted mutagenesis its the 'misc_difference', i.e. the second option of the two you present. This is also true in your second scenario because it is changes possibly unintentional, possibly random mutagenesis / or just unintentional errors during cloning. Finally, this is also true for SOME of the 'errors' you are reporting in comments:

for the first i, ii and iii that appears to be just correcting stuff - administration, just cross-check with those submitting.
for 'variations' the second i and ii in comments, that falls into the 'misc_difference', because a form of mutagenesis, albeit probably unintentional.


Conclusion
The 'variation' feature description isn't clear given the description you reported against NCBI's official Helpdesk response. Its a fair question, its a clear answer, but doesn't explain why the description is confusing.

On 'errors' of mis-identification, e.g. correctly identified e.g. via local Blast or Blat, e.g. sequenced the wrong target, I would use an internal marker, e.g. returned to contributor for comment. Pass it back to the contributor and ask for their input and depending what they say simply mark the record in the 'Comments section' e.g. originally defined as XX. The contributor could have made all sorts of different errors, e.g. annotation - copied and pasted the wrong stuff - experimental anything and you need to give them a chance to respond. Uploading these records on an external database, i.e. Genbank, needs a joint agreement with all parties concerned because it is certainly valid information for someone.

Note, the official blurb here, doesn't appear informative:

Disrupted genes and gene fragments
Sometimes a genome will have adjacent or nearby genes that seem to be
only part of a protein. In many cases these indicate a possible
problem with the sequence and/or annotation. A related issue is the
presence of internal stop codons in the conceptual translation of a
CDS that looks like it should be a real CDS. These problems may be due
to a variety of reasons, including mutations or sequencing or assembly
artifacts. They can be annotated in a number of ways:
Annotate the gene with /pseudo to indicate that there is a problem
with the gene. Note that this qualifier does NOT mean that the gene is
a pseudogene. (see point 2, below, if it is known that the gene IS a
pseudogene) If multiple gene fragments were present initially, then
add a single gene feature which covers all of the potential coding
regions and add the pseudo qualifier. If known, a note qualifier may
be added indicating why this gene is disrupted, for example:

 1        200     gene
                         gene    phoA
                         gene_desc     alkaline phosphatase
                         locus_tag     OBB_0001
                         pseudo
                         note    nonfunctional due to frameshift


---

### Answer by username_0 on 2022-04-24. Score: 1

Overview
To properly address the questions, each possible case must be listed:

Case 1: divergence between sequences (sequence in the file vs sequence of the molecule); two cases:
a- (wrong) sequence in the file vs (correct) sequence in the molecule
b- ("correct", theoretical) sequence in the file vs (actual, mutated) sequence in the molecule

Case 2: divergence between a feature annotation in the file vs the correct ("true" or reference) biological feature; three cases:
a- wrong location of the feature
b- wrong feature name
c- variant sequence of the feature


Short answer
Case 1a: The sequence in the GenBank file must be corrected and the version number of the file must be incremented (see Q2).
Case 1b: A new GenBank file consistent with the actual molecule must be created and the misc_difference feature key must be used to annotate the variations where relevant.
Cases 2a and 2b: Feature information in the GenBank file must be corrected and the Modification Date of the file must be updated (see Q2).
Case 2c: The misc_difference feature must be used to annotate the variation and the Modification Date of the file must be updated if necessary (see Q2).
Long answer
Q1. How to handle the situation when there are divergences between a GenBank file and an actual or theoretical molecule?
The misc_difference feature key
First, let's analyse the situation with the misc_difference feature key.
According to the INSDC documentation:
Feature Key           misc_difference


Definition            feature sequence is different from that presented 
                      in the entry and cannot be described by any other 
                      difference key (old_sequence, variation, or modified_base);

Optional qualifiers   /allele="text"
                      /clone="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text" 
                      /note="text"
                      /old_locus_tag="text" (single token)
                      /phenotype="text"
                      /replace="text" 
                      /standard_name="text"

Comment               the misc_difference feature key should be used to 
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                      use /replace="" to annotate deletion, e.g. 
                      misc_difference 412..433
                                      /replace=""  

From the Definition above, there is still some ambiguity as to what situation is described. Indeed, "feature sequence is different from that presented in the entry" can describe both of the following cases:

the sequence in the file and/or in the molecule is not the one expected for the feature annotated

the sequence in the molecule is not the one represented in the file


Note: In fact, as we will see below, there should be no divergence between the sequence in the file and the sequence of the molecule.
As for the Comment that says that "the misc_difference feature key should be used to describe variability that arises as a result of genetic manipulation (e.g. site directed mutagenesis)", it does not really clarify which of the two cases above is the one intended. Further, the example given in the Comment, which at first sight seems more enlightening, still carries some ambiguity. It says that to annotate a deletion, one should use:
misc_difference 412..433
                /replace=""

All in all, it implies that the deletion is in the molecule (vs in the represented sequence), that it arose as a result of genetic manipulation and is therefore indicated in the file as explained above.
However, it is very counter-intuitive, and not very practical when searching sequences of variants among sequence files, to have a sequence file that is supposed to describe a molecule and in which the sequence is actually different from the sequence encountered in the molecule it is supposed to describe.
And indeed, this view goes against common observed recent practice where the misc_difference feature key is used to indicate how to go from the sequence represented in the sequence file to another version:

Example 1: https://www.ncbi.nlm.nih.gov/nuccore/D90147.1
     misc_difference 308..310
                     /gene="mucA"
                     /note="conflict: deletion"
                     /citation=[1]
                     /replace=""

The indicated deletion is not in the sequence but is a way to go from the sequence to a previously reported CDS sequence for the annotated gene.
Translation of the CDS sequence in the GenBank file:
MKVDIFESSGASRVHSIPFYLQRISAGFPSPAQGYEKQELNLHEYCVRHPSATYFLRVSGSSMEDGRIHDGDVLVVDRSLTASHGSIVVACIHNEFTVKRLLLRPRPCLMPMNKDFPVYYIDPDNESVEIWGVVTHSLIEHPVCLR*

Previously reported CDS (see: https://www.ncbi.nlm.nih.gov/nuccore/M13388;
hyphen/dash in the sequence added by me to mark the difference)
MKVDIFESSGAS-VHSIPFYLQRISAGFPSPAQGYEKQELNLHEYCVRHPSATYFLRVSGSSMEDGRIHDGDVLVVDRSLTASHGSIVVACIHNEFTVKRLLLRPRPCLMPMNKDFPVYYIDPDNESVEIWGVVTHSLIEHPVCLR*

Ref.: https://pubmed.ncbi.nlm.nih.gov/1902211/

Example 2
In each of the GenBank files below, the misc_difference feature key is used to indicate how to go from the (mutated) sequence in the GenBank file to the sequence of the "wild type".

Escherichia coli uidA gene for beta-glucuronidase mutant E413G, clone M2
https://www.ncbi.nlm.nih.gov/nuccore/AM180563.1
 misc_difference 1238
               /gene="uidA"
               /experiment="experimental evidence, no additional details
               recorded"
               /note="site directed mutagenesis"
               /compare=S69414.1
               /allele="wild type"
               /phenotype="Active GUS"
               /replace="a"


Escherichia coli uidA gene for beta-glucuronidase mutant C464R, clone M4
https://www.ncbi.nlm.nih.gov/nuccore/AM180564.1
misc_difference 1390
                /gene="uidA"
                /experiment="experimental evidence, no additional details
                recorded"
                /note="site directed mutagenesis"
                /compare=S69414.1
                /allele="wild type"
                /phenotype="Active GUS"
                /replace="t"



See also:

Escherichia coli uidA gene for beta-glucuronidase mutant Y468C, clone M6
https://www.ncbi.nlm.nih.gov/nuccore/AM180565.1

Escherichia coli uidA gene for beta-glucuronidase mutant W549R, clone M12
https://www.ncbi.nlm.nih.gov/nuccore/AM180566.1



Example 3: https://www.ncbi.nlm.nih.gov/nuccore/KC999035.4
 misc_difference 137414..137415

                 /note="TT compared with single T in original ISCfr1
                 sequence that would result in two partial transposase
                 proteins. Confirmed as correct in raw sequence data."

Here again the misc_difference feature key is used to describe how to pass from the represented sequence to a previous sequence version of the considered gene.


So the misc_difference feature key is appropriate when the sequence in the sequence file does match the sequence in the molecule but does not (any more) match another reference sequence (possibly referred to by another gene-related feature) because variations in the sequence have occurred due to some genetic manipulation or, as in Example 3, due to discrepancy with previous sequencing results.
Thus, the misc_difference feature key is appropriate in Cases 1b and 2c, and the /replace qualifier should be used to indicate how to get from the represented sequence to the other reference sequence for the designated feature (in Example 3 above, the /note qualifier is used instead to describe the difference).
Other cases
There are three other cases left:

Case 1a: the features in the GenBank file are consistent with the molecule, but the sequence in the file is wrong
Cases 2a and 2b: the sequence in the GenBank file is consistent with the molecule, but information about some features is wrong

Those three cases can all be classified as a GenBank file containing errors (in contrast with case 1b where the "error" is in the obtained molecule and with case 2c where the information in the GenBank file is merely incomplete). A GenBank file containing errors is just not appropriate, and the solution here is simply to correct the GenBank file to accurately describe the corresponding molecule.
All that remains is to answer to the second question about how to track changes...
Q2. How to keep track of changes between versions of a GenBank file when corrections or updates are made?
Versioning and time-stamping GenBank files
GenBank files have two fields that can be used to track their history: the Modification Date field and the VERSION field.
The VERSION field uses an accession.version format, where the accession number is fixed and the version number is incremented only when the sequence data is modified.
See:

https://www.ncbi.nlm.nih.gov/genbank/samplerecord/
https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#ModificationDateB
https://www.ncbi.nlm.nih.gov/genbank/samplerecord/#VersionB

Note that only modifications in the sequence result in a change in the version number. Changes in annotations only affect the timestamp (Modification Date) of the file.
See:

https://www.ncbi.nlm.nih.gov/genbank/sequenceids/

With this information we can now describe how to properly keep track of the successive versions of a GenBank file in each of the cases described in Q1.
Proper way to keep track of modifications in GenBank files
Case 1a: (wrong) sequence in the file vs (correct) sequence in the molecule
The sequence in the file must be corrected and the version number of the file must be incremented by one.
Moreover the respective file versions (the former and the newer) must refer to each other in the COMMENT field of their respective header.
See:

https://www.ncbi.nlm.nih.gov/genbank/release/current/ --> 3.4.2  Entry Organization

Case 1b: ("correct", theoretical) sequence in the file vs (actual, mutated) sequence in the molecule
A new GenBank file and new molecule name must be created to accurately describe the variant molecule. The misc_difference feature key along with the /replace qualifier must be used as described above where it is deemed appropriate.
The gene-related features affected should keep the same /gene qualifier, but an /allele qualifier might be used to indicate the presence of a difference and the /note and /phenotype qualifiers can be used to indicate the reference sequence to which the allele should be compared to and any possible phenotypic consequence of the variation.
The misc_difference feature key can be given the same gene-related qualifier information as the gene where the difference occurs (/gene, /allele, /note...)
In the special case of variants unintentionally obtained during genetic construction or genetic manipulation, an altered name for the molecule and GenBank file might be used to reflect the fact that the corresponding sequence is different from the theoretically expected sequence, and the misc_difference feature key should be used to indicate any difference that occurred during the construction compared to the initially targetted sequence.
Cases 2a, 2b or 2c: wrong or incomplete feature data
The feature annotation in the file must be corrected and the Modification Date field must be updated according to the correction date.
Additional references
Sequence revision history

https://www.ncbi.nlm.nih.gov/genbank/sequencerevisionhistory/

Examples of revision history of some GenBank files

https://www.ncbi.nlm.nih.gov/nuccore/U46667?report=girevhist
https://www.ncbi.nlm.nih.gov/nuccore/NM_029716?report=girevhist
https://www.ncbi.nlm.nih.gov/nuccore/NM_033116?report=girevhist

Added for convenience
Here below is reproduced the description and usage of some feature keys and qualifiers that might be relevant to, or linked with, the question of how to describe and annotate sequence variants.
I grouped those definitions there as it might help one to distinguish the different types of variations and divergences and to understand and chose the appropriate term to annotate sequence variants.
Selected feature keys
gene
Feature Key           gene


Definition            region of biological interest identified as a gene 
                      and for which a name has been assigned;

Optional qualifiers   /allele="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /function="text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text"
                      /note="text"
                      /old_locus_tag="text" (single token)
                      /operon="text"
                      /product="text"
                      /pseudo
                      /pseudogene="TYPE"
                      /phenotype="text"
                      /standard_name="text"
                      /trans_splicing


Comment               the gene feature describes the interval of DNA that 
                      corresponds to a genetic trait or phenotype; the feature is,
                      by definition, not strictly bound to it's positions at the 
                      ends;  it is meant to represent a region where the gene is 
                      located.

misc_difference
Feature Key           misc_difference


Definition            feature sequence is different from that presented 
                      in the entry and cannot be described by any other 
                      difference key (old_sequence, variation, or modified_base);

Optional qualifiers   /allele="text"
                      /clone="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text" 
                      /note="text"
                      /old_locus_tag="text" (single token)
                      /phenotype="text"
                      /replace="text" 
                      /standard_name="text"

Comment               the misc_difference feature key should be used to 
                      describe variability that arises as a result of 
                      genetic manipulation (e.g. site directed mutagenesis);
                      use /replace="" to annotate deletion, e.g. 
                      misc_difference 412..433
                                      /replace=""  

modified_base
Feature Key           modified_base


Definition            the indicated nucleotide is a modified nucleotide and
                      should be substituted for by the indicated molecule
                      (given in the mod_base qualifier value)
 
Mandatory qualifiers  /mod_base=

Optional qualifiers   /allele="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /frequency="text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text"
                      /note="text"
                      /old_locus_tag="text" (single token)

Comment               value is limited to the restricted vocabulary for 
                      modified base abbreviations;

old_sequence
Feature Key           old_sequence


Definition            the presented sequence revises a previous version of the
                      sequence at this location;

Mandatory qualifiers  /citation=[number]
                      Or
                      /compare=[accession-number.sequence-version]

Optional qualifiers   /allele="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text"
                      /note="text"
                      /old_locus_tag="text" (single token)
                      /replace="text"

Comment               /replace="" is used to annotate deletion, e.g. 
                      old_sequence 12..15
                      /replace="" 
                      NOTE: This feature key is not valid in entries/records
                      created from 15-Oct-2007.

variation
Feature Key           variation

Definition            a related strain contains stable mutations from the same
                      gene (e.g., RFLPs, polymorphisms, etc.) which differ
                      from the presented sequence at this location (and
                      possibly others);

Optional qualifiers   /allele="text"
                      /db_xref=":"
                      /experiment="[CATEGORY:]text"
                      /frequency="text"
                      /gene="text"
                      /gene_synonym="text"
                      /inference="[CATEGORY:]TYPE[(same species)][:EVIDENCE_BASIS]"
                      /locus_tag="text" (single token)
                      /map="text"
                      /note="text"
                      /old_locus_tag="text" (single token)
                      /phenotype="text"
                      /product="text"
                      /replace="text"
                      /standard_name="text"

Comment               used to describe alleles, RFLP's,and other naturally
                      occurring mutations and  polymorphisms; variability arising
                      as a result of genetic manipulation (e.g. site directed
                      mutagenesis) should be described with the misc_difference
                      feature; use /replace="" to annotate deletion, e.g. 
                      variation   4..5
                                  /replace=""  

Selected qualifiers
/allele
  Qualifier       /allele=
  Definition      name of the allele for the given gene 
  Value format    "text"
  Example         /allele="adh1-1"
  Comment         all gene-related features (exon, CDS etc) for a given 
                  gene should share the same /allele qualifier value; 
                  the /allele qualifier value must, by definition, be 
                  different from the /gene qualifier value; when used with 
                  the variation feature key, the allele qualifier value 
                  should be that of the variant.

/clone
  Qualifier       /clone=
  Definition      clone from which the sequence was obtained
  Value format    "text"
  Example         /clone="lambda-hIL7.3"
  Comment         not more than one clone should be specified for a given source 
                  feature;  to indicate that the sequence was obtained from
                  multiple clones, multiple source features should be given.

/compare
  Qualifier       /compare=
  Definition      Reference details of an existing public INSD entry 
                  to which a comparison is made
  Value format    [accession-number.sequence-version]
  Example         /compare=AJ634337.1
  Comment         not to be used for new entries from 14-MAY-2021; This qualifier
                  may be used on the following features: misc_difference, unsure,
                  old_sequence and variation.
                  The feature "old_sequence" must have either a
                  /citation or a /compare qualifier. Multiple /compare
                  qualifiers with different contents are allowed within a 
                  single feature. 
                  This qualifier is not intended for large-scale annotation 
                  of variations, such as SNPs.

/function
  Qualifier       /function=
  Definition      function attributed to a sequence
  Value format    "text"
  Example         function="essential for recognition of cofactor"
  Comment         /function is used when the gene name and/or product name do not 
                  convey the function attributable to a sequence.

/gene
  Qualifier       /gene=
  Definition      symbol of the gene corresponding to a sequence region
  Value format    "text"
  Example         /gene="ilvE" 

/locus_tag
  Qualifier       /locus_tag=
  Definition      a submitter-supplied, systematic, stable identifier for a gene
                  and its associated features, used for tracking purposes
  Value Format    "text"(single token) 
                  but not "<1-5 letters><5-9 digit integer>[.]"
  Example         /locus_tag="ABC_0022" 
                  /locus_tag="A1C_00001"
  Comment         /locus_tag can be used with any feature that /gene can be used
                  with; identical /locus_tag values may be used within an
                  entry/record, but only if the identical /locus_tag values are 
                  associated with the same gene; in all other circumstances the
                  /locus_tag value must be unique within that entry/record.
                  Multiple /locus_tag values are not allowed within one feature for
                  entries created after 15-OCT-2004. 
                  If a /locus_tag needs to be re-assigned the /old_locus_tag
                  qualifier should be used to store the old value. The /locus_tag
                  value should not be in a format which resembles INSD accession
                  numbers, accession.version, or /protein_id identifiers.

/map
  Qualifier       /map=
  Definition      genomic map position of feature
  Value format    "text"
  Example         /map="8q12-q13"

/note
  Qualifier       /note=
  Definition      any comment or additional information
  Value format    "text"
  Example         /note="This qualifier is equivalent to a comment."

/operon
  Qualifier       /operon=
  Definition      name of the group of contiguous genes transcribed into a 
                  single transcript to which that feature belongs.
  Value format    "text"
  Example         /operon="lac"

/phenotype
  Qualifier       /phenotype=
  Definition      phenotype conferred by the feature, where phenotype is defined as
                  a physical, biochemical or behavioural characteristic or set of 
                  characteristics
  Value format    "text"
  Example         /phenotype="erythromycin resistance"

/product
  Qualifier       /product=
  Definition      name of the product associated with the feature, e.g. the mRNA of
                  an mRNA feature, the polypeptide of a CDS, the mature peptide of
                  a mat_peptide, etc.
  Value format    "text"
  Example         /product="trypsinogen" (when qualifier appears in CDS feature)
                  /product="trypsin" (when qualifier appears in mat_peptide
                  feature)
                  /product="XYZ neural-specific transcript" (when qualifier appears
                  in mRNA feature)

/pseudo
  Qualifier       /pseudo
  Definition      indicates that this feature is a non-functional version of the
                  element named by the feature key
  Value format    none
  Example         /pseudo
  Comment         The qualifier /pseudo should be used to describe non-functional 
                  genes that are not formally described as pseudogenes, e.g. CDS 
                  has no translation due to other reasons than pseudogenisation
                  events.
                  Other reasons may include sequencing or assembly errors.
                  In order to annotate pseudogenes the qualifier /pseudogene= must
                  be used indicating the TYPE which can be taken from the INSDC
                  controlled vocabulary for pseudogenes.

/pseudogene
  Qualifier       /pseudogene=
  Definition      indicates that this feature is a pseudogene of the element
                  named by the feature key
  Value format    "TYPE"
                  where TYPE is one of the following:
                  processed, unprocessed, unitary, allelic, unknown

  Example         /pseudogene="processed"
                  /pseudogene="unprocessed"
                  /pseudogene="unitary"
                  /pseudogene="allelic"
                  /pseudogene="unknown"

  Comment         TYPE is a term taken from the INSDC controlled vocabulary for
                  pseudogenes (http://www.insdc.org/documents/pseudogene-
                  qualifier-vocabulary):

                  processed: the pseudogene has arisen by reverse transcription
                  of a mRNA into cDNA, followed by reintegration into the genome.
                  Therefore, it has lost any intron/exon structure, and it might
                  have a pseudo-polyA-tail.

                  unprocessed: the pseudogene has arisen from a copy of the
                  parent gene by duplication followed by accumulation of random
                  mutations.
                  The changes, compared to their functional homolog, include
                  insertions, deletions, premature stop codons, frameshifts and a
                  higher proportion of non-synonymous versus synonymous
                  substitutions.
                  
                  unitary: the pseudogene has no parent. It is the original gene,
                  which is functional is some species but disrupted in some way
                  (indels, mutation, recombination) in another species or strain.

                  allelic: a (unitary) pseudogene that is stable in the
                  population but importantly it has a functional alternative
                  allele also in the population. i.e., one strain may have the
                  gene, another strain may have the pseudogene.
                  MHC haplotypes have allelic pseudogenes.

                  unknown: the submitter does not know the method of
                  pseudogenisation.

/standard_name
  Qualifier       /standard_name=
  Definition      accepted standard name for this feature
  Value format    "text"
  Example         /standard_name="dotted"
  Comment         use /standard_name to give full gene name, but use /gene to
                  give gene symbol (in the above example /gene="Dt").


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