Picard gives empty value for HS_LIBRARY_SIZE when running CollectHsMetrics

Question

[Bertalan_Takacs]

Basically what the title says. I have samples from human gene panel sequencing. I aligned them the human genome and ran CollectHsMetrics. I didn't receive any error messages and the output looks normal overall, except for the HS_LIBRARY_SIZE value which appears to be empty. I am using the newest human genome version on ensembl and a target list file generated in-house. I tried several different interval list versions and reference genomes but the HS_LIBRARY_SIZE remains empty. What else can I try? What could cause this problem?

An example output:

## htsjdk.samtools.metrics.StringHeader
# Started on: Tue Mar 11 13:36:57 CET 2025

## METRICS CLASS    picard.analysis.directed.HsMetrics BAIT_SET BAIT_TERRITORY  BAIT_DESIGN_EFFICIENCY  ON_BAIT_BASES   NEAR_BAIT_BASES OFF_BAIT_BASES  PCT_SELECTED_BASES  PCT_OFF_BAIT    ON_BAIT_VS_SELECTED MEAN_BAIT_COVERAGE  PCT_USABLE_BASES_ON_BAIT    PCT_USABLE_BASES_ON_TARGET  FOLD_ENRICHMENT HS_LIBRARY_SIZE HS_PENALTY_10X  HS_PENALTY_20X  HS_PENALTY_30X  HS_PENALTY_40X  HS_PENALTY_50X  HS_PENALTY_100X TARGET_TERRITORY    GENOME_SIZE TOTAL_READS PF_READS    PF_BASES    PF_UNIQUE_READS PF_UQ_READS_ALIGNED PF_BASES_ALIGNED    PF_UQ_BASES_ALIGNED ON_TARGET_BASES PCT_PF_READS    PCT_PF_UQ_READS PCT_PF_UQ_READS_ALIGNED MEAN_TARGET_COVERAGE    MEDIAN_TARGET_COVERAGE  MAX_TARGET_COVERAGE MIN_TARGET_COVERAGE ZERO_CVG_TARGETS_PCT    PCT_EXC_DUPE    PCT_EXC_ADAPTER PCT_EXC_MAPQ    PCT_EXC_BASEQ   PCT_EXC_OVERLAP PCT_EXC_OFF_TARGET  FOLD_80_BASE_PENALTY    PCT_TARGET_BASES_1X PCT_TARGET_BASES_2X PCT_TARGET_BASES_10X    PCT_TARGET_BASES_20X    PCT_TARGET_BASES_30X    PCT_TARGET_BASES_40X    PCT_TARGET_BASES_50X    PCT_TARGET_BASES_100X   PCT_TARGET_BASES_250X   PCT_TARGET_BASES_500X   PCT_TARGET_BASES_1000X  PCT_TARGET_BASES_2500X  PCT_TARGET_BASES_5000X  PCT_TARGET_BASES_10000X PCT_TARGET_BASES_25000X PCT_TARGET_BASES_50000X PCT_TARGET_BASES_100000XAT_DROPOUT  GC_DROPOUT  HET_SNP_SENSITIVITY HET_SNP_Q   SAMPLE  LIBRARY READ_GROUP Celemics-All-Cancer-55_2023-04-11    194756  1   1709889627  428313074   2419515882  0.469139    0.530861    0.799685    8779.650573 0.295277    0.163358    5971.129562     0   0   0   0   0   0   194756  3099750718  38349644    38349644    5790796244  38349644    38276748    4557718583  4557718583  945974613   1   1   0.998099    4857.229626 4488.5  14811   121 0   0   0.000725    0.090316    0.007106    0.377153    0.317199    1.657758    1   1   1   1   1   1   1   1   0.999415    0.998644    0.995482    0.873786    0.402026    0.033524    0   0   0   12.04638    0.121985    1   -1

[GenoMax]

These are paired-end sequence data?

[Bertalan_Takacs]

Yes, they are

Answer 1

[Bertalan_Takacs]

Nevermind, I've figured it out. The issue was that I didn't run MarkDuplicates before CollectHsMetrics