Picard CalculateHsMetrics perTargetCoverage for Novaseq bams

Question

[genya35]

Hello, I would like to use Picard's CalculateHsMetrics to calculate per target coverage for Novaseq bam files. It seems that the tool is not able to calculate mean/normalized coverage for Novaseq bams but works well with Hiseq bams. Novaseq bams report quality scores differently, therefore, my guess this is what is causing the issue. Do you have any suggestions.

java -jar /usr/local/bin/picard.jar CalculateHsMetrics \
BAIT_INTERVALS=targets.interval_list \
TARGET_INTERVALS=targets.interval_list \
INPUT=/alignT/test_clipped_overlaps.bam \
OUTPUT=/alignT/QC/test.HsMetrics.out \
REFERENCE_SEQUENCE=/ref/GRCh37/genome/human_g1k_v37.fasta \
PER_TARGET_COVERAGE=/alignT/QC/test.PerTargetCoverage.txt \
VALIDATION_STRINGENCY=SILENT \
METRIC_ACCUMULATION_LEVEL=ALL_READS \
VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false

Thanks

[GenoMax]

tool is not able to calculate mean/normalized coverage for Novaseq bams

NovaSeq uses binned Q scores but they should still be normal BAM's. What do you mean by the tool is not able to calculate coverage?

[genya35]

It returns 0s in these two columns.

[GenoMax]

So the command is working. It is not producing a logical answer you are expecting. Are chromosome names matching in all files. That would be the first place to start checking.