<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>

CC BY-SA 3.0
2018-02-15T21:01:43.187Z
thankyu !! what are those nUmber of secondary and non-unique alignments, ambiguous, and no feature aligned?
beginner
CC BY-SA 3.0
2018-02-15T21:59:24.000Z
And what are the causes reads genomic origin - Intergenic shows higher number? sequencing error, mapping error, unannotated genes? I see in some of the post that for human genome, it's very likely cz of unannotated gens. The annotation of a genome could never be finished, as it could never be sequenced completely. Is this right?
beginner
CC BY-SA 3.0
2018-02-15T23:20:37.067Z
@raju I've updated my reply.
Devon Ryan
CC BY-SA 3.0
2018-02-16T08:29:19.057Z
Thanks for the answers. I have to tell you that for "alignment" with HISAT2 I didn't use any gtf file. I used only standard genome index from HISAT2 website. I used gencode new version (gencode.v27 with both protein and non-coding genes) gtf for Stringtie tool. In the above qualimap result I didn't mention the paired-end option "-pe". So, I have rerun the qualimap again with "-pe" and also "-p strand-specific-reverse" which is RF in HISAT2. The following are the results.
beginner
CC BY-SA 3.0
2018-02-16T08:30:11.897Z
Reads alignment

    reads aligned (left/right) = 86,704,161 / 85,485,519
    read pairs aligned  = 83,397,732
    total alignments = 244,302,519
    secondary alignments = 72,112,839
    non-unique alignments = 22,973,098
    aligned to genes  = 249
    ambiguous alignments = 25
    no feature assigned = 9,759
    not aligned = 5,887,822; Reads genomic origin

    exonic =  249 (2.49%)
    intronic = 693 (6.92%)
    intergenic = 9,066 (90.59%)
    overlapping exon = 54 (0.54%)


Transcript coverage profile

    5' bias = 0.39
    3' bias = 10.04
    5'-3' bias = �
beginner
CC BY-SA 3.0
2018-02-15T20:34:33.810Z
2018-02-15T23:19:37.523Z
2018-02-15T23:19:37.523Z
Devon Ryan
<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>

<rna-seq><sam><phylogenetics><rna>
Alignment QC differences between HISAT2 and Qualimap
# Alignment QC differences between HISAT2 and Qualimap

(Asked by: username_0 on 2018-02-15)

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.

**Tags:** rna-seq, sam, phylogenetics, rna

---

## 1 answers

### Answer by username_1 on 2018-02-15. Score: 2

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](http://qualimap.bioinfo.cipf.es/doc_html/analysis.html#id7) 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.

> **Comments:**
> **username_0**: thankyu !! what are those nUmber of secondary and non-unique alignments, ambiguous, and no feature aligned?  
> **username_0**: And what are the causes reads genomic origin - Intergenic shows higher number? sequencing error, mapping error, unannotated genes? I see in some of the post that for human genome, it's very likely cz of unannotated gens. The annotation of a genome could never be finished, as it could never be sequenced completely. Is this right?  
> **username_1**: @raju I've updated my reply.  
> **username_0**: Thanks for the answers. I have to tell you that for "alignment" with HISAT2 I didn't use any gtf file. I used only standard genome index from HISAT2 website. I used gencode new version (gencode.v27 with both protein and non-coding genes) gtf for Stringtie tool. In the above qualimap result I didn't mention the paired-end option "-pe". So, I have rerun the qualimap again with "-pe" and also "-p strand-specific-reverse" which is RF in HISAT2. The following are the results.  
> **username_0**: Reads alignment      reads aligned (left/right) = 86,704,161 / 85,485,519     read pairs aligned  = 83,397,732     total alignments = 244,302,519     secondary alignments = 72,112,839     non-unique alignments = 22,973,098     aligned to genes  = 249     ambiguous alignments = 25     no feature assigned = 9,759     not aligned = 5,887,822; Reads genomic origin      exonic =  249 (2.49%)     intronic = 693 (6.92%)     intergenic = 9,066 (90.59%)     overlapping exon = 54 (0.54%)   Transcript coverage profile      5' bias = 0.39     3' bias = 10.04     5'-3' bias = �  

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