All sequenced reads in piared end sequencing don't have mate pairs?
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13 months ago
piyushjo ▴ 560

Hi,

I am using the new HISAT2 v2.2.0 to perform alignment of paired end RNA-seq data. The alignment report suggests that not all the reads were paired ended. I am slightly confused by this. I thought all reads in PE sequencing have mate pairs. Thew new HISAT2 summary looks like this.

HISAT2 summary stats:

    Total pairs: 76700832
            Aligned concordantly or discordantly 0 time: 3686808 (4.81%)
            Aligned concordantly 1 time: 60966514 (79.49%)
            Aligned concordantly >1 times: 11843091 (15.44%)
            Aligned discordantly 1 time: 204419 (0.27%)
    Total unpaired reads: 7373616
            Aligned 0 time: 3934307 (53.36%)
            Aligned 1 time: 2579590 (34.98%)
            Aligned >1 times: 859719 (11.66%)
    Overall alignment rate: 97.44%
  

While the old HISAT2 summary, used to look like this (taken from HISAT2 website)

Alignment summary (not for the same data, just want to show it used to say 100% of reads were paired end)

10000 reads; of these:   10000 (100.00%) were paired; of these:
    650 (6.50%) aligned concordantly 0 times
    8823 (88.23%) aligned concordantly exactly 1 time
    527 (5.27%) aligned concordantly >1 times
    ----
    650 pairs aligned concordantly 0 times; of these:
      34 (5.23%) aligned discordantly 1 time
    ----
    616 pairs aligned 0 times concordantly or discordantly; of these:
      1232 mates make up the pairs; of these:
        660 (53.57%) aligned 0 times
        571 (46.35%) aligned exactly 1 time

        1 (0.08%) aligned >1 times
96.70% overall alignment rate

I calculated that now almost 9% of my reads are not paired end. Is that normal? Did older HISAT2 used to discard unpaired reads for alignment?

HISAT2 paired end sequencing • 360 views
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Did you manipulate the fastq files somehow? Like trimming or any custom kind of filtering?

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No. I just ran fasqtc before aligning and din't perform any trimming. The second summary is actually not for the same data. I will clarify that in the question.

This unrelated post also has an example where the read summary is separated into "paired end reads" and "unpaired end reads".

hisat2 --sra-acc with paired reads producing single read output

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13 months ago
h.mon 32k

You should run the same sample with both versions of hisat and then compare the summary output, it would be less confusing. It seems to me just a change in terminology in the summary output: whereas in hisat 2.2.0 there is a "section" called unpaired reads:

Total unpaired reads: 7373616
        Aligned 0 time: 3934307 (53.36%)
        Aligned 1 time: 2579590 (34.98%)
        Aligned >1 times: 859719 (11.66%)

Previously, in hisat 2.1.0, corresponds to this section:

616 pairs aligned 0 times concordantly or discordantly; of these:
  1232 mates make up the pairs; of these:
    660 (53.57%) aligned 0 times
    571 (46.35%) aligned exactly 1 time
    1 (0.08%) aligned >1 times

The term unpaired read wasn't explicitly used, but the same information was present in the summary output.

Again, you will get a better picture by comparing the summary of the same sample, instead of comparing two different samples, each run with a different version of hisat.

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Thanks! I will confirm this.

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I checked that in v2.1 of HISAT2, the section with XXX pairs aligned 0 times concordantly or discordantly, is similar to HISAT2 v2.2, where the reads are unpaired. Thanks for your explanation!

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