text: user1369: I used hisat2 for aligning reads to to the genome. I have an alignment summary for sample1 as follows:
  89038751 reads; of these:
    89038751 (100.00%) were paired; of these:
      5641019 (6.34%) aligned concordantly 0 times
      67665552 (76.00%) aligned concordantly exactly 1 time
      15732180 (17.67%) aligned concordantly >1 times
      ----
      5641019 pairs aligned concordantly 0 times; of these:
        651415 (11.55%) aligned discordantly 1 time
      ----
      4989604 pairs aligned 0 times concordantly or discordantly; of these:
        9979208 mates make up the pairs; of these:
          5887822 (59.00%) aligned 0 times
          2656902 (26.62%) aligned exactly 1 time
          1434484 (14.37%) aligned >1 times
  96.69% overall alignment rate
  
  To check the alignment quality I used Qualimap. In the report I see the following:
  Reads alignment
  
  Number of mapped reads: 172,189,680
  Total number of alignments: 244,302,519
  Number of secondary alignments: 72,112,839
  Number of non-unique alignments:    22,973,098
  Aligned to genes:   2,081
  Ambiguous alignments:   173
  No feature assigned:    178,975
  Not aligned:    5,887,822
  
  Reads genomic origin
  
  Exonic: 2,081 / 1.15%
  Intronic:   6,866 / 3.79%
  Intergenic: 172,109 / 95.06%
  Intronic/intergenic overlapping exon:   302 / 0.17%
  
  Transcript coverage profile
  
  5' bias:    0.69
  3' bias:    2.85
  5'-3' bias: 0.39
  
  Can anyone tell me why the numbers look different? Did I go wrong somewhere? Help me in understanding the results.
  
  user77: 89038751 pairs times 2 is 178 million reads. That times the approximately 97% alignment rate is around 172 million reads, which is what qualimap is reporting.
  A "non-unique" alignment is one that can map to multiple places (either equally well, or where all of the alignments are of similar enough quality). Such alignments have a single "primary" alignment and one or more "secondary" alignments, which is where the "secondary" metrics come from.
  For the qualimap output, there are additional metrics describing how reads overlap genes (or not). Of note are the "ambiguous" alignments, which are those overlapping multiple genes. "No feature" metrics, then, refer to alignments not overlapping any gene (see the qualimap documentation for further details).
  My guess is that you have such a high "intergenic" rate because you've either used the wrong GTF file or have told qualimap the wrong strand setting. For unusual organisms one expects many reads to arise from unannotated genes. This is not the case for common model organisms, such as mice and humans, where there are very few remaining commonly expressed unannotated genes.
  
original_text: 1369: <p>I used hisat2 for aligning reads to to the genome. I have an alignment summary for sample1 as follows:</p>
  
  <pre><code>89038751 reads; of these:
    89038751 (100.00%) were paired; of these:
      5641019 (6.34%) aligned concordantly 0 times
      67665552 (76.00%) aligned concordantly exactly 1 time
      15732180 (17.67%) aligned concordantly &gt;1 times
      ----
      5641019 pairs aligned concordantly 0 times; of these:
        651415 (11.55%) aligned discordantly 1 time
      ----
      4989604 pairs aligned 0 times concordantly or discordantly; of these:
        9979208 mates make up the pairs; of these:
          5887822 (59.00%) aligned 0 times
          2656902 (26.62%) aligned exactly 1 time
          1434484 (14.37%) aligned &gt;1 times
  96.69% overall alignment rate
  </code></pre>
  
  <p>To check the alignment quality I used Qualimap. In the report I see the following:</p>
  
  <pre><code>Reads alignment
  
  Number of mapped reads: 172,189,680
  Total number of alignments: 244,302,519
  Number of secondary alignments: 72,112,839
  Number of non-unique alignments:    22,973,098
  Aligned to genes:   2,081
  Ambiguous alignments:   173
  No feature assigned:    178,975
  Not aligned:    5,887,822
  
  Reads genomic origin
  
  Exonic: 2,081 / 1.15%
  Intronic:   6,866 / 3.79%
  Intergenic: 172,109 / 95.06%
  Intronic/intergenic overlapping exon:   302 / 0.17%
  
  Transcript coverage profile
  
  5' bias:    0.69
  3' bias:    2.85
  5'-3' bias: 0.39
  </code></pre>
  
  <p>Can anyone tell me why the numbers look different? Did I go wrong somewhere? Help me in understanding the results.</p>
   77: <p>89038751 pairs times 2 is 178 million reads. That times the approximately 97% alignment rate is around 172 million reads, which is what qualimap is reporting.</p>
  
  <p>A "non-unique" alignment is one that can map to multiple places (either equally well, or where all of the alignments are of similar enough quality). Such alignments have a single "primary" alignment and one or more "secondary" alignments, which is where the "secondary" metrics come from.</p>
  
  <p>For the qualimap output, there are additional metrics describing how reads overlap genes (or not). Of note are the "ambiguous" alignments, which are those overlapping multiple genes. "No feature" metrics, then, refer to alignments not overlapping any gene (see <a href="http://qualimap.bioinfo.cipf.es/doc_html/analysis.html#id7" rel="nofollow noreferrer">the qualimap documentation</a> for further details).</p>
  
  <p>My guess is that you have such a high "intergenic" rate because you've either used the wrong GTF file or have told qualimap the wrong strand setting. For unusual organisms one expects many reads to arise from unannotated genes. This is not the case for common model organisms, such as mice and humans, where there are very few remaining commonly expressed unannotated genes.</p>
  