Why difference in Hisat2 and bamqc from qualimap results?

Question

[Vasu]

Hi,

I'm using hisat2 for aligning reads to the genome. For a few samples I see some differences by using hisat2 and bamqc from qualimap.

Hisat2 output:

37317546 reads; of these:
  37317546 (100.00%) were paired; of these:
    14771091 (39.58%) aligned concordantly 0 times
    7081700 (18.98%) aligned concordantly exactly 1 time
    15464755 (41.44%) aligned concordantly >1 times
    ----
    14771091 pairs aligned concordantly 0 times; of these:
      1186424 (8.03%) aligned discordantly 1 time
    ----
    13584667 pairs aligned 0 times concordantly or discordantly; of these:
      27169334 mates make up the pairs; of these:
        22785681 (83.87%) aligned 0 times
        1973892 (7.27%) aligned exactly 1 time
        2409761 (8.87%) aligned >1 times
69.47% overall alignment rate

For the same sample using bam file "qualimap bamqc results" are as following:

Reference

 number of bases = 3,099,750,718 bp
 number of contigs = 194


Globals

 number of windows = 593

 number of reads = 202,671,876
 number of mapped reads = 179,886,195 (88.76%)

 number of mapped paired reads (first in pair) = 90,666,939
 number of mapped paired reads (second in pair) = 89,219,256
 number of mapped paired reads (both in pair) = 171,622,685
 number of mapped paired reads (singletons) = 8,263,510
 number of mapped bases = 30,000,606,541 bp
 number of sequenced bases = 8,238,989,876 bp
 number of aligned bases = 0 bp
 number of duplicated reads (estimated) = 95,761,476
 duplication rate = 25.6%


 Insert size

 mean insert size = 29,714.41
 std insert size = 464,081.65
 median insert size = 1199


Mapping quality

 mean mapping quality = 13.82


ACTG content

 number of A's = 1,679,640,440 bp (20.39%)
 number of C's = 2,133,982,067 bp (25.9%)
 number of T's = 1,805,802,126 bp (21.92%)
 number of G's = 2,619,565,243 bp (31.79%)
 number of N's = 0 bp (0%)

 GC percentage = 57.7%


 Mismatches and indels

general error rate = 0
number of mismatches = 32,158,659
number of insertions = 876,201
mapped reads with insertion percentage = 0.49%
number of deletions = 174,885
mapped reads with deletion percentage = 0.1%
homopolymer indels = 24.62%

In hisat2 output I see overall alignment rate is 69.47% and bamqc results I see number of mapped reads is 88%. Which is right one?

[Carlo Yague]

Both metrics are right. In your bam file, you have 88% of mapped reads. From your input reads, only 69% are mapped (once or more than once). The mutlitple alignments are causing the difference

[Vasu]

Ok. And how can I get unmapped reads percentage? 88% of mapped reads is once?

[Carlo Yague]

The percentage of unmapped reads (compared to the total number of reads) is 30.53%.

The percentage of unmapped reads (compared to the total number of alignments in the bam file) is 12%.

[Vasu]

Thank you. But could you please tell me how this total number of reads and total number of alignments are different? And could you also tell me how u calculated the above percentages.

[Carlo Yague]

But could you please tell me how this total number of reads and total number of alignments are different?

Because for one read, there can be more than one alignment : (41.44%) aligned concordantly >1 times

And could you also tell me how u calculated the above percentages.

100% - 69.47% = 30.53% (1 - (number of reads that map at least once/total number of reads) = proportion of unmapped reads)

100% - 88% = 12% (1 - (number of effective alignments/total number of entries in the bam file) = proportion of unmapped reads in the bam file)

[Vasu]

Thank you very much. I guess there is a typo in ur comment. It should be 100% - 88% = 12%.