# Alignment QC differences between HISAT2 and Qualimap

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

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.

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.

• thankyu !! what are those nUmber of secondary and non-unique alignments, ambiguous, and no feature aligned? Feb 15 '18 at 21:01
• 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? Feb 15 '18 at 21:59
• @raju I've updated my reply. Feb 15 '18 at 23:20
• 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. Feb 16 '18 at 8:29
• 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 = � Feb 16 '18 at 8:30