How to interpret flagstat output

Question

[deniselavezzari]

Hi, I have an issue similar to some published.

FastQC gives me 2,941,170 Total sequence, whereas the flagstat outputs these:

3510466 + 0 in total (QC-passed reads + QC-failed reads)
3508788 + 0 primary
0 + 0 secondary
1678 + 0 supplementary
0 + 0 duplicates
0 + 0 primary duplicates
3510466 + 0 mapped (100.00% : N/A)
3508788 + 0 primary mapped (100.00% : N/A)
3508788 + 0 paired in sequencing
1755007 + 0 read1
1753781 + 0 read2
3501708 + 0 properly paired (99.80% : N/A)
3505324 + 0 with itself and mate mapped
3464 + 0 singletons (0.10% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

I cannot undestand the difference which is not explained by supplementary or unmapped reads. Thanks a lot.

Answer 1

[Istvan Albert]

It sums up like this:

3508788 +1678 = 3510466

there are no unmapped reads, some of your reads generate more than one alignment, these are the supplementary (chimeric) alignments. 1678 such supplementary alignments are present.

The data seems to have been filtered though, some pairs are broken, the unmapped mate is not present.

[deniselavezzari]

Thanks a lot Istvan! Even with these supplementary, I still cannot explain the higher number with respect to FastQC results.

[Istvan Albert]

I totally missed the FastQC number which seems to be the main point of the question. I focused on flagstat alone.

I don't what to say about that - the numbers should match, perhaps you running it on a different version of the file