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

Question

[avp25]

Hello there,

I am trying to use the --sra-acc function from hisat2 with paired end data. I have installed both hisat2 and the sra-toolkit successfully. Indeed, the mapping works fine but the SAM output shows reads mapped as if they were single reads. My hisat2 command looks like:

hisat2  --no-mixed --no-discordant -x ../ref//hg38/genome --sra-acc <accession> -S output.sam

where <accession> is a single number, and this SRA accession links to both 1.fastq.gz and 2.fastq.gz

Is there any way to tell hisat2 that the accession refers to paired end reads?

Thanks!

Anna

[h.mon]

Check if the SRA data really contain paired-end reads. I have seen single-end data marked as paired-end - in that case, it was a faulty upload. But bottom-line is don't trust SRA blindly.

[GenoMax]

Paired-end reads are aligned together. Many aligners drop the read designations from read name and may encode that information in SAM flags (83 and 147).

[Nitin Narwade]

I really don't know about hisat2 --sra-acc option, but while downloading the data using NCBI-SRA Toolkit I had the same issue. fastq-dump utility of NCBI-SRA Toolkit gave me single fastq file for paired entry. This issue has been solved by --split-files option provided by fastq-dump.

Answer 1

[poojasethiya]

For single end SRA data hisat2 gives summary statistics as:

HISAT2 summary stats:
Total reads: 27870948
                 Aligned 0 time: 11196555 (40.17%)
                 Aligned 1 time: 15284312 (54.84%)
                 Aligned >1 times: 1390081 (4.99%)
         Overall alignment rate: 59.83%

For paired end SRA data hisat2 gives summary statistics as:

HISAT2 summary stats:
        Total pairs: 9113937
                Aligned concordantly or discordantly 0 time: 4233427 (46.45%)
                Aligned concordantly 1 time: 4493249 (49.30%)
                Aligned concordantly >1 times: 351445 (3.86%)
                Aligned discordantly 1 time: 35816 (0.39%)
        Total unpaired reads: 8466854
                Aligned 0 time: 6882022 (81.28%)
                Aligned 1 time: 1428012 (16.87%)
                Aligned >1 times: 156820 (1.85%)
        Overall alignment rate: 62.24%

From these results it can be seen that hisat2 treats paired-end and single end data differently.