99.9% read mapped by flagstat but zero coverage calculated
Entering edit mode
21 months ago
godth13teen ▴ 70

Hi, I have downloaded and aligned PacBio HIFI NA12878 sample against GRCh38 genome, using Minimap2.

java -jar $PICARD CollectWgsMetrics I=NA12878_CCS_sorted.bam O=collect_wgs_metrics.txt R=$ref_fasta

Then I use picard collectwgsmetrics to calculate the coverage, I found that the coverage is 0.0. Then I used samtools flagstat and got this result:

$ samtools flagstate NA12878_CCS_sorted.bam

14410787 + 0 in total (QC-passed reads + QC-failed reads)
4878353 + 0 secondary
450400 + 0 supplementary
0 + 0 duplicates
14396335 + 0 mapped (99.90% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

It seems like all of the reads are mapped, but how come they have zero coverage? I also checked the alignment manually, and found many read have 0 flag (this one is very subjective).

How did those 2 tools have quite different result? And what is the correct way to calculate coverage of long read sample?

alignment coverage • 573 views
Entering edit mode

Please post the command line of picard and the output. Zero (the flag) means mapped as single-end read, not unmapped, or are you referring to MAPQ?

Entering edit mode

Oh right, so I misinterpreted the flag of those reads. I also updated the command in my post

Entering edit mode

You could quickly put your BAM into qualimap to see what this one outputs. It gives a nice graphic output with the number of bp covered at least x-fold.


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