Answers.Body: <p>In one of its possible output modes, kraken2 will give you read-level and kmer-level information about how it came to its conclusions about taxonomic assignment. You've provided a lot of detail in your question about your process of generating the data, and the taxon assignment table, but I expect your specific question will best be answered by digging deeper into your own data.</p>
  <p>To explain a bit further, individual reads are assigned to a taxon based on the kmer assignments for that read (i.e. each kmer within a read essentially votes for the read taxon). The behaviour of assignment after changing the database is unpredictable at a read level because the kmer assignments will change. Even if diversity is increased, that increased diversity will alter the &quot;votes&quot; from the kmers, and could result in a particular taxon crossing a threshold (and increasing in apparent abundance) because other taxa now have fewer votes.</p>
  <p>In any case, Kraken2 is known to assign taxa in a way that is not necessarily a good representation of the actual content. If you want to make statements about the proportion of different taxa in your samples, I'd recommend that you try out using Bracken on your Kraken2 results. It will correct some assignments based on sequence similarities in the database:</p>
  <blockquote>
  <p>Bracken is a companion program to Kraken 1, KrakenUniq, or Kraken 2 While Kraken classifies reads to multiple levels in the taxonomic tree, Bracken allows estimation of abundance at a single level using those classifications (e.g. Bracken can estimate abundance of species within a sample).</p>
  </blockquote>
  <p><a href="https://github.com/jenniferlu717/Bracken?tab=readme-ov-file#step-1-generate-the-bracken-database-file-databasexmerskmer_distrib" rel="nofollow noreferrer">https://github.com/jenniferlu717/Bracken?tab=readme-ov-file#step-1-generate-the-bracken-database-file-databasexmerskmer_distrib</a></p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-07-27T21:01:50.507Z
Answers.Text: Thanks for the ref to bracken. You wrote "I expect your specific question will best be answered by digging deeper into your own data.". Note that the puzzling observation I made is actually not dependent on my data, since it seems to stem from some effect at database construction.
Answers.UserDisplayName: bli
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-07-28T01:11:55.903Z
Answers.Text: @bli I've added a bit more detail to explain my reasoning. The main thing is that database changes lead to unpredictable effects. You can't always assume that class assignment fractions will go down when database diversity is increased. That's why it's important to use tools like Bracken that take this kmer weirdness into account.
Answers.UserDisplayName: gringer
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-07-26T22:11:45.567Z
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Answers.LastEditDate: 2024-07-28T04:18:27.793Z
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Body: <p>I built a protozoa kraken 2 database (&quot;db1&quot;), from which I further built two other databases (&quot;db2&quot; and &quot;db3&quot;) by adding extra genomes in the Leishmania sub-genus<sup>*</sup>.</p>
  <p>When comparing results obtained when analyzing sequencing data using these databases, I noticed a strange increase in the number of reads assigned to the Leishmania chagasi species (which is a terminal node in the taxonomy for both databases) when going from db2 to db3.</p>
  <p>According to my superficial understanding of kraken, I didn't expect this to happen, because <strong>db3 was built using a higher diversity of genomes outside the L. chagasi species than db2</strong>. If I expected any effect, this should have reduced the number of k-mers that are found specifically in L. chagasi, because some of these k-mers in db2 could have been present in the new genomes added to db3.</p>
  <p>This increase in analysis reports parallels an increase in the number of minimizers as reported by <code>kraken2-inspect</code>. Below is a table comparing the <code>kraken2-inspect</code> reports for db2 and db3.</p>
  <p>I only show the lines corresponding to Leishmania subgenus and its parents.</p>
  <p>Compared to the <code>kraken2-inspect</code> output, I removed the taxid and re-ordered columns, for readability. <code>nb_min</code> and <code>nb_min_lvl</code> correspond to the number of minimizers reported on columns 2 and 3 of the <code>kraken2-inspect</code> default output (See <a href="https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown#inspecting-a-kraken-2-databases-contents" rel="nofollow noreferrer">https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown#inspecting-a-kraken-2-databases-contents</a>).</p>
  <p>The line for L. chagasi is in bold, and shows <strong>a substantial increase (more than 20-fold)</strong> from 63874 minimizers in db2 to 1389504 in db3:</p>
  <div class="s-table-container"><table class="s-table">
  <thead>
  <tr>
  <th>taxname</th>
  <th style="text-align: right;">lvl</th>
  <th style="text-align: right;">% (db2)</th>
  <th style="text-align: right;">nb_min (db2)</th>
  <th style="text-align: right;">nb_min_lvl (db2)</th>
  <th style="text-align: right;">% (db3)</th>
  <th style="text-align: right;">nb_min (db3)</th>
  <th style="text-align: right;">nb_min_lvl (db3)</th>
  </tr>
  </thead>
  <tbody>
  <tr>
  <td>root</td>
  <td style="text-align: right;">R</td>
  <td style="text-align: right;">100.0</td>
  <td style="text-align: right;">228330589</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">100</td>
  <td style="text-align: right;">254009353</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>cellular organisms</td>
  <td style="text-align: right;">R1</td>
  <td style="text-align: right;">100.0</td>
  <td style="text-align: right;">228330589</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">100</td>
  <td style="text-align: right;">254009353</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Eukaryota</td>
  <td style="text-align: right;">D</td>
  <td style="text-align: right;">100.0</td>
  <td style="text-align: right;">228330589</td>
  <td style="text-align: right;">4685</td>
  <td style="text-align: right;">100</td>
  <td style="text-align: right;">254009353</td>
  <td style="text-align: right;">6669</td>
  </tr>
  <tr>
  <td>Discoba</td>
  <td style="text-align: right;">D1</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Euglenozoa</td>
  <td style="text-align: right;">P</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Kinetoplastea</td>
  <td style="text-align: right;">C</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Metakinetoplastina</td>
  <td style="text-align: right;">C1</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Trypanosomatida</td>
  <td style="text-align: right;">O</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Trypanosomatidae</td>
  <td style="text-align: right;">F</td>
  <td style="text-align: right;">23.4</td>
  <td style="text-align: right;">53431766</td>
  <td style="text-align: right;">1645</td>
  <td style="text-align: right;">31.14</td>
  <td style="text-align: right;">79110530</td>
  <td style="text-align: right;">2029</td>
  </tr>
  <tr>
  <td>Leishmaniinae</td>
  <td style="text-align: right;">F1</td>
  <td style="text-align: right;">19.87</td>
  <td style="text-align: right;">45358915</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">27.97</td>
  <td style="text-align: right;">71037679</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania</td>
  <td style="text-align: right;">G</td>
  <td style="text-align: right;">19.87</td>
  <td style="text-align: right;">45358915</td>
  <td style="text-align: right;">259764</td>
  <td style="text-align: right;">27.97</td>
  <td style="text-align: right;">71037679</td>
  <td style="text-align: right;">305088</td>
  </tr>
  <tr>
  <td>Leishmania</td>
  <td style="text-align: right;">G1</td>
  <td style="text-align: right;">14.67</td>
  <td style="text-align: right;">33503508</td>
  <td style="text-align: right;">4587885</td>
  <td style="text-align: right;">23.3</td>
  <td style="text-align: right;">59182271</td>
  <td style="text-align: right;">12771940</td>
  </tr>
  <tr>
  <td>Leishmania donovani species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">3.61</td>
  <td style="text-align: right;">8243592</td>
  <td style="text-align: right;">6215217</td>
  <td style="text-align: right;">5.01</td>
  <td style="text-align: right;">12729716</td>
  <td style="text-align: right;">1378715</td>
  </tr>
  <tr>
  <td>Leishmania donovani</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">0.63</td>
  <td style="text-align: right;">1439307</td>
  <td style="text-align: right;">1439307</td>
  <td style="text-align: right;">3.71</td>
  <td style="text-align: right;">9428437</td>
  <td style="text-align: right;">8055955</td>
  </tr>
  <tr>
  <td>Leishmania donovani Ld 2001</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">0.48</td>
  <td style="text-align: right;">1207432</td>
  <td style="text-align: right;">1207432</td>
  </tr>
  <tr>
  <td>Leishmania donovani Ld 39</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">0.06</td>
  <td style="text-align: right;">165050</td>
  <td style="text-align: right;">165050</td>
  </tr>
  <tr>
  <td><strong>Leishmania chagasi</strong></td>
  <td style="text-align: right;"><strong>S</strong></td>
  <td style="text-align: right;"><strong>0.03</strong></td>
  <td style="text-align: right;"><strong>63874</strong></td>
  <td style="text-align: right;"><strong>63874</strong></td>
  <td style="text-align: right;"><strong>0.55</strong></td>
  <td style="text-align: right;"><strong>1389504</strong></td>
  <td style="text-align: right;"><strong>1389504</strong></td>
  </tr>
  <tr>
  <td>Leishmania infantum</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">0.23</td>
  <td style="text-align: right;">525194</td>
  <td style="text-align: right;">520206</td>
  <td style="text-align: right;">0.21</td>
  <td style="text-align: right;">533060</td>
  <td style="text-align: right;">528882</td>
  </tr>
  <tr>
  <td>Leishmania infantum JPCM5</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;">0.0</td>
  <td style="text-align: right;">4988</td>
  <td style="text-align: right;">4988</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">4178</td>
  <td style="text-align: right;">4178</td>
  </tr>
  <tr>
  <td>Leishmania mexicana species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">4.39</td>
  <td style="text-align: right;">10033887</td>
  <td style="text-align: right;">4875993</td>
  <td style="text-align: right;">4.18</td>
  <td style="text-align: right;">10626363</td>
  <td style="text-align: right;">5076482</td>
  </tr>
  <tr>
  <td>Leishmania amazonensis</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">1.22</td>
  <td style="text-align: right;">2782684</td>
  <td style="text-align: right;">2782684</td>
  <td style="text-align: right;">1.23</td>
  <td style="text-align: right;">3123079</td>
  <td style="text-align: right;">3123079</td>
  </tr>
  <tr>
  <td>Leishmania mexicana</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">1.04</td>
  <td style="text-align: right;">2375210</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">0.96</td>
  <td style="text-align: right;">2426802</td>
  <td style="text-align: right;">2191283</td>
  </tr>
  <tr>
  <td>Leishmania mexicana MHOM/GT/2001/U1103</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;">1.04</td>
  <td style="text-align: right;">2375210</td>
  <td style="text-align: right;">2375210</td>
  <td style="text-align: right;">0.09</td>
  <td style="text-align: right;">235519</td>
  <td style="text-align: right;">235519</td>
  </tr>
  <tr>
  <td>Leishmania martiniquensis</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">3.44</td>
  <td style="text-align: right;">8749402</td>
  <td style="text-align: right;">8749402</td>
  </tr>
  <tr>
  <td>Leishmania major species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">2.49</td>
  <td style="text-align: right;">5691012</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">2.35</td>
  <td style="text-align: right;">5980712</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania major</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">2.49</td>
  <td style="text-align: right;">5691012</td>
  <td style="text-align: right;">5634913</td>
  <td style="text-align: right;">2.35</td>
  <td style="text-align: right;">5980712</td>
  <td style="text-align: right;">5350171</td>
  </tr>
  <tr>
  <td>Leishmania major strain LV39c5</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">0.17</td>
  <td style="text-align: right;">419323</td>
  <td style="text-align: right;">419323</td>
  </tr>
  <tr>
  <td>Leishmania major strain SD 75.1</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">0.08</td>
  <td style="text-align: right;">192440</td>
  <td style="text-align: right;">192440</td>
  </tr>
  <tr>
  <td>Leishmania major strain Friedlin</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;">0.02</td>
  <td style="text-align: right;">56099</td>
  <td style="text-align: right;">56099</td>
  <td style="text-align: right;">0.01</td>
  <td style="text-align: right;">18778</td>
  <td style="text-align: right;">18778</td>
  </tr>
  <tr>
  <td>Leishmania tropica species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">1.89</td>
  <td style="text-align: right;">4803669</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania tropica</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">1.89</td>
  <td style="text-align: right;">4803669</td>
  <td style="text-align: right;">4796954</td>
  </tr>
  <tr>
  <td>Leishmania tropica L590</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;"></td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">6715</td>
  <td style="text-align: right;">6715</td>
  </tr>
  <tr>
  <td>Leishmania aethiopica species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">2.17</td>
  <td style="text-align: right;">4947132</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">1.39</td>
  <td style="text-align: right;">3520469</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania aethiopica</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">2.17</td>
  <td style="text-align: right;">4947132</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">1.39</td>
  <td style="text-align: right;">3520469</td>
  <td style="text-align: right;">3057668</td>
  </tr>
  <tr>
  <td>Leishmania aethiopica L147</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;">2.17</td>
  <td style="text-align: right;">4947132</td>
  <td style="text-align: right;">4947132</td>
  <td style="text-align: right;">0.18</td>
  <td style="text-align: right;">462801</td>
  <td style="text-align: right;">462801</td>
  </tr>
  <tr>
  <td>Viannia</td>
  <td style="text-align: right;">G1</td>
  <td style="text-align: right;">5.08</td>
  <td style="text-align: right;">11595643</td>
  <td style="text-align: right;">5588978</td>
  <td style="text-align: right;">4.55</td>
  <td style="text-align: right;">11550320</td>
  <td style="text-align: right;">5553385</td>
  </tr>
  <tr>
  <td>Leishmania braziliensis species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">1.33</td>
  <td style="text-align: right;">3037436</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">1.19</td>
  <td style="text-align: right;">3032336</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania braziliensis</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">1.33</td>
  <td style="text-align: right;">3037436</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">1.19</td>
  <td style="text-align: right;">3032336</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania braziliensis MHOM/BR/75/M2904</td>
  <td style="text-align: right;">S1</td>
  <td style="text-align: right;">1.33</td>
  <td style="text-align: right;">3037436</td>
  <td style="text-align: right;">3037436</td>
  <td style="text-align: right;">1.19</td>
  <td style="text-align: right;">3032336</td>
  <td style="text-align: right;">3032336</td>
  </tr>
  <tr>
  <td>Leishmania guyanensis species complex</td>
  <td style="text-align: right;">G2</td>
  <td style="text-align: right;">1.3</td>
  <td style="text-align: right;">2969229</td>
  <td style="text-align: right;">0</td>
  <td style="text-align: right;">1.17</td>
  <td style="text-align: right;">2964599</td>
  <td style="text-align: right;">0</td>
  </tr>
  <tr>
  <td>Leishmania panamensis</td>
  <td style="text-align: right;">S</td>
  <td style="text-align: right;">1.3</td>
  <td style="text-align: right;">2969229</td>
  <td style="text-align: right;">2969229</td>
  <td style="text-align: right;">1.17</td>
  <td style="text-align: right;">2964599</td>
  <td style="text-align: right;">2964599</td>
  </tr>
  </tbody>
  </table></div>
  <p>My question: <strong>How is such an increase possible, when the only genomes added are outside the clade?</strong></p>
  <p>I don't know if this can help understanding what happens, but L. chagasi could actually be considered a sub-clade of L. infantum (chagasi is the descendant of infantum brought to the New world by European conquistadores), but in the NCBI taxonomy used to build the databases, they are sister S-level taxa within the G2 &quot;L. donovani species complex&quot; (together with the L. donovani species, that has two S1-level strains added in db3 compared to db2).</p>
  <p><sup>*</sup> Here is how I built the databases</p>
  <pre><code>kraken2-build --download-taxonomy --db protozoa
  kraken2-build --download-library protozoa --db protozoa
  cp -r protozoa db2
  cp -r protozoa db3
  mv protozoa db1
  kraken2-build --build --threads 16 --db db1
  # Download genomes
  # Add genomes to db2 using `kraken2-build --add-to-library &lt;genome&gt;/*.fna --db db2`
  kraken2-build --build --threads 16 --db db2
  # Add even more genomes do db3 using `kraken2-build --add-to-library &lt;genome&gt;/*.fna --db db3`
  kraken2-build --build --threads 16 --db db3
  </code></pre>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-07-28T02:31:50.470Z
Comments.Text: I will look at this question over the next week. There's clearly a lot going on here. What I need to think about is "outside the clade" ... I'm not sure about that.
Comments.UserDisplayName: M__
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-07-29T13:20:31.107Z
Comments.Text: To elaborate: If I had added genomes belonging to L. chagasi (for instance, at the S1 level), it would not have surprised me to see a larger number of k-mers associated to L. chagasi at the species level. New genomes should give a better idea of the diversity observable within a given clade. However, with genomes only added elsewhere, I don't get it. Maybe something to do with the distinction between k-mers and minimizers?
Comments.UserDisplayName: bli
Tags: <metagenome><k-mer><kraken>
Title: How adding genomes to a kraken database can increase the number of minimizers associated with a leaf of the taxonomy?
ThreadText: # How adding genomes to a kraken database can increase the number of minimizers associated with a leaf of the taxonomy?
  
  (Asked by: username_0 on 2024-07-26)
  
  I built a protozoa kraken 2 database ("db1"), from which I further built two other databases ("db2" and "db3") by adding extra genomes in the Leishmania sub-genus\*.
  
  When comparing results obtained when analyzing sequencing data using these databases, I noticed a strange increase in the number of reads assigned to the Leishmania chagasi species (which is a terminal node in the taxonomy for both databases) when going from db2 to db3.
  
  According to my superficial understanding of kraken, I didn't expect this to happen, because **db3 was built using a higher diversity of genomes outside the L. chagasi species than db2**. If I expected any effect, this should have reduced the number of k-mers that are found specifically in L. chagasi, because some of these k-mers in db2 could have been present in the new genomes added to db3.
  
  This increase in analysis reports parallels an increase in the number of minimizers as reported by `kraken2-inspect`. Below is a table comparing the `kraken2-inspect` reports for db2 and db3.
  
  I only show the lines corresponding to Leishmania subgenus and its parents.
  
  Compared to the `kraken2-inspect` output, I removed the taxid and re-ordered columns, for readability. `nb_min` and `nb_min_lvl` correspond to the number of minimizers reported on columns 2 and 3 of the `kraken2-inspect` default output (See <https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown#inspecting-a-kraken-2-databases-contents>).
  
  The line for L. chagasi is in bold, and shows **a substantial increase (more than 20-fold)** from 63874 minimizers in db2 to 1389504 in db3:
  
  | taxname | lvl | % (db2) | nb\_min (db2) | nb\_min\_lvl (db2) | % (db3) | nb\_min (db3) | nb\_min\_lvl (db3) |
  | --- | --- | --- | --- | --- | --- | --- | --- |
  | root | R | 100.0 | 228330589 | 0 | 100 | 254009353 | 0 |
  | cellular organisms | R1 | 100.0 | 228330589 | 0 | 100 | 254009353 | 0 |
  | Eukaryota | D | 100.0 | 228330589 | 4685 | 100 | 254009353 | 6669 |
  | Discoba | D1 | 23.4 | 53431766 | 0 | 31.14 | 79110530 | 0 |
  | Euglenozoa | P | 23.4 | 53431766 | 0 | 31.14 | 79110530 | 0 |
  | Kinetoplastea | C | 23.4 | 53431766 | 0 | 31.14 | 79110530 | 0 |
  | Metakinetoplastina | C1 | 23.4 | 53431766 | 0 | 31.14 | 79110530 | 0 |
  | Trypanosomatida | O | 23.4 | 53431766 | 0 | 31.14 | 79110530 | 0 |
  | Trypanosomatidae | F | 23.4 | 53431766 | 1645 | 31.14 | 79110530 | 2029 |
  | Leishmaniinae | F1 | 19.87 | 45358915 | 0 | 27.97 | 71037679 | 0 |
  | Leishmania | G | 19.87 | 45358915 | 259764 | 27.97 | 71037679 | 305088 |
  | Leishmania | G1 | 14.67 | 33503508 | 4587885 | 23.3 | 59182271 | 12771940 |
  | Leishmania donovani species complex | G2 | 3.61 | 8243592 | 6215217 | 5.01 | 12729716 | 1378715 |
  | Leishmania donovani | S | 0.63 | 1439307 | 1439307 | 3.71 | 9428437 | 8055955 |
  | Leishmania donovani Ld 2001 | S1 |  |  |  | 0.48 | 1207432 | 1207432 |
  | Leishmania donovani Ld 39 | S1 |  |  |  | 0.06 | 165050 | 165050 |
  | **Leishmania chagasi** | **S** | **0.03** | **63874** | **63874** | **0.55** | **1389504** | **1389504** |
  | Leishmania infantum | S | 0.23 | 525194 | 520206 | 0.21 | 533060 | 528882 |
  | Leishmania infantum JPCM5 | S1 | 0.0 | 4988 | 4988 | 0 | 4178 | 4178 |
  | Leishmania mexicana species complex | G2 | 4.39 | 10033887 | 4875993 | 4.18 | 10626363 | 5076482 |
  | Leishmania amazonensis | S | 1.22 | 2782684 | 2782684 | 1.23 | 3123079 | 3123079 |
  | Leishmania mexicana | S | 1.04 | 2375210 | 0 | 0.96 | 2426802 | 2191283 |
  | Leishmania mexicana MHOM/GT/2001/U1103 | S1 | 1.04 | 2375210 | 2375210 | 0.09 | 235519 | 235519 |
  | Leishmania martiniquensis | S |  |  |  | 3.44 | 8749402 | 8749402 |
  | Leishmania major species complex | G2 | 2.49 | 5691012 | 0 | 2.35 | 5980712 | 0 |
  | Leishmania major | S | 2.49 | 5691012 | 5634913 | 2.35 | 5980712 | 5350171 |
  | Leishmania major strain LV39c5 | S1 |  |  |  | 0.17 | 419323 | 419323 |
  | Leishmania major strain SD 75.1 | S1 |  |  |  | 0.08 | 192440 | 192440 |
  | Leishmania major strain Friedlin | S1 | 0.02 | 56099 | 56099 | 0.01 | 18778 | 18778 |
  | Leishmania tropica species complex | G2 |  |  |  | 1.89 | 4803669 | 0 |
  | Leishmania tropica | S |  |  |  | 1.89 | 4803669 | 4796954 |
  | Leishmania tropica L590 | S1 |  |  |  | 0 | 6715 | 6715 |
  | Leishmania aethiopica species complex | G2 | 2.17 | 4947132 | 0 | 1.39 | 3520469 | 0 |
  | Leishmania aethiopica | S | 2.17 | 4947132 | 0 | 1.39 | 3520469 | 3057668 |
  | Leishmania aethiopica L147 | S1 | 2.17 | 4947132 | 4947132 | 0.18 | 462801 | 462801 |
  | Viannia | G1 | 5.08 | 11595643 | 5588978 | 4.55 | 11550320 | 5553385 |
  | Leishmania braziliensis species complex | G2 | 1.33 | 3037436 | 0 | 1.19 | 3032336 | 0 |
  | Leishmania braziliensis | S | 1.33 | 3037436 | 0 | 1.19 | 3032336 | 0 |
  | Leishmania braziliensis MHOM/BR/75/M2904 | S1 | 1.33 | 3037436 | 3037436 | 1.19 | 3032336 | 3032336 |
  | Leishmania guyanensis species complex | G2 | 1.3 | 2969229 | 0 | 1.17 | 2964599 | 0 |
  | Leishmania panamensis | S | 1.3 | 2969229 | 2969229 | 1.17 | 2964599 | 2964599 |
  
  My question: **How is such an increase possible, when the only genomes added are outside the clade?**
  
  I don't know if this can help understanding what happens, but L. chagasi could actually be considered a sub-clade of L. infantum (chagasi is the descendant of infantum brought to the New world by European conquistadores), but in the NCBI taxonomy used to build the databases, they are sister S-level taxa within the G2 "L. donovani species complex" (together with the L. donovani species, that has two S1-level strains added in db3 compared to db2).
  
  \* Here is how I built the databases
  
  ```
  kraken2-build --download-taxonomy --db protozoa
  kraken2-build --download-library protozoa --db protozoa
  cp -r protozoa db2
  cp -r protozoa db3
  mv protozoa db1
  kraken2-build --build --threads 16 --db db1
  # Download genomes
  # Add genomes to db2 using `kraken2-build --add-to-library <genome>/*.fna --db db2`
  kraken2-build --build --threads 16 --db db2
  # Add even more genomes do db3 using `kraken2-build --add-to-library <genome>/*.fna --db db3`
  kraken2-build --build --threads 16 --db db3
  
  ```
  
  **Tags:** metagenome, k-mer, kraken
  
  > **Comments:**
  > **username_1**: I will look at this question over the next week. There's clearly a lot going on here. What I need to think about is "outside the clade" ... I'm not sure about that.  
  > **username_0**: To elaborate: If I had added genomes belonging to L. chagasi (for instance, at the S1 level), it would not have surprised me to see a larger number of k-mers associated to L. chagasi at the species level. New genomes should give a better idea of the diversity observable within a given clade. However, with genomes only added elsewhere, I don't get it. Maybe something to do with the distinction between k-mers and minimizers?  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_2 on 2024-07-26. Score: 1
  
  In one of its possible output modes, kraken2 will give you read-level and kmer-level information about how it came to its conclusions about taxonomic assignment. You've provided a lot of detail in your question about your process of generating the data, and the taxon assignment table, but I expect your specific question will best be answered by digging deeper into your own data.
  
  To explain a bit further, individual reads are assigned to a taxon based on the kmer assignments for that read (i.e. each kmer within a read essentially votes for the read taxon). The behaviour of assignment after changing the database is unpredictable at a read level because the kmer assignments will change. Even if diversity is increased, that increased diversity will alter the "votes" from the kmers, and could result in a particular taxon crossing a threshold (and increasing in apparent abundance) because other taxa now have fewer votes.
  
  In any case, Kraken2 is known to assign taxa in a way that is not necessarily a good representation of the actual content. If you want to make statements about the proportion of different taxa in your samples, I'd recommend that you try out using Bracken on your Kraken2 results. It will correct some assignments based on sequence similarities in the database:
  
  > Bracken is a companion program to Kraken 1, KrakenUniq, or Kraken 2 While Kraken classifies reads to multiple levels in the taxonomic tree, Bracken allows estimation of abundance at a single level using those classifications (e.g. Bracken can estimate abundance of species within a sample).
  
  <https://github.com/jenniferlu717/Bracken?tab=readme-ov-file#step-1-generate-the-bracken-database-file-databasexmerskmer_distrib>
  
  > **Comments:**
  > **username_0**: Thanks for the ref to bracken. You wrote "I expect your specific question will best be answered by digging deeper into your own data.". Note that the puzzling observation I made is actually not dependent on my data, since it seems to stem from some effect at database construction.  
  > **username_2**: @username_0 I've added a bit more detail to explain my reasoning. The main thing is that database changes lead to unpredictable effects. You can't always assume that class assignment fractions will go down when database diversity is increased. That's why it's important to use tools like Bracken that take this kmer weirdness into account.  
  
  ---
  
  