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Curated variation benchmarks for challenging medically relevant autosomal genes


The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

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Fig. 1: GIAB developed a process to create new phased small variant and SV benchmarks for 273 CMRGs.
Fig. 2: The new CMRG benchmark contains more challenging variants and regions than previous benchmarks.
Fig. 3: The new benchmark covers the gene SMN1, which was previously excluded due to mapping challenges for all technologies in the highly identical segmental duplication.
Fig. 4: The benchmark resolves the gene CBS, which has a highly homologous gene (CBSL) due to a false duplication in GRCh38 that is not in HG002 or GRCh37.
Fig. 5: The new CMRG small variant benchmark includes more challenging variants and identifies more false negatives in a standard short-read callset (Illumina–BWA-MEM–GATK) than the previous v4.2.1 benchmark in these challenging genes.

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Data availability

The PacBio HiFi reads used to generate the hifiasm assembly for the benchmark are in the NCBI Sequence Read Archive with accession numbers SRR10382245, SRR10382244, SRR10382249, SRR10382248, SRR10382247 and SRR10382246. The v1.00 benchmark VCF and BED files, as well as Liftoff gene annotations, assembly–assembly alignments and variant calls, are available at, and as a DOI at This is released as a separate benchmark from v4.2.1, because it includes a small fraction of the genome, it has different characteristics from the mapping-based v4.2.1 and v4.2.1 only includes small variants. Using v4.2.1 and the CMRG benchmarks as two separate benchmarks enables users to obtain broader performance metrics for most of the genome and for a small set of particularly challenging genes, respectively. The masked GRCh38 reference, recently updated to v2 with additional false duplications from the Telomere-to-Telomere Consortium, is under We recommend using v3.0 GA4GH/GIAB stratification bed files intended for use with when benchmarking, which are available at These stratifications include bed files corresponding to false duplications and collapsed duplications in GRCh38. All data have no restrictions, as the HG002 sample has an open consent from the Personal Genome Project.

Code availability

Scripts used to develop the CMRG benchmark and generate figures and tables for the manuscript are available at The previously developed assembly, which was used as the basis of this benchmark, was from hifiasm v0.11.

A variety of open source software was used for variant calling for the evaluations of the benchmark, including NextDenovo2.2-beta.0, DRAGEN 3.6.3, NeuSomatic’s submission for the PrecisionFDA truth challenge v2 (ref. 12) (BWA-MEM50 version 0.7.17-r1188 ( and GATK version gatk- (, Parabricks_DeepVariant (Parabricks Pipelines DeepVariant v3.0.0_2 (, Sentieon (DNAscope) version sentieon_release_201911 (, BWA-MEM and Strelka2 (BWA-MEM version 0.7.17-r1188 ( and Strelka2 version 2.9.10 (, BWA-MEM50(v0.7.8), Picard tools ( (ver. 1.83), GATK52 (v3.4-0), GATK (v3.5), BWA-MEM v0.7.15-r1140, SAMtools53 v1.3, Picard v2.10.10, GATK v3.8, DELLY54 v0.8.5, GRIDSS55 v2.9.4, LUMPY56 v0.3.1, Manta57 v1.6.0, Wham58 v1.7.0, NanoPlot60 v1.27.0, Filtlong v0.2.0, minimap2 (refs. 40,60) v2.17-r941, cuteSV v1.0.8, Sniffles61 v1.0.12, SURVIVOR59 v1.0.7, BWA v0.7.15, GATK v3.6, Java v1.8.0_74 (OpenJDK), Picard Tools v2.6.0, Sambamba63 v0.6.7, Samblaster64 v0.1.24, Samtools v1.9, DeepVariant v1.0 and Liftoff32 v1.4.0.


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We thank the Genome Reference Consortium for their curation efforts of GRCh37 and GRCh38 (, especially V.A. Schneider and P.A. Kitts from the National Institutes of Health (NIH)/NCBI for developing the falsely duplicated regions that should be masked in GRCh38. We thank S. Miller at NIST for helping make available benchmark sets and READMEs. Certain commercial equipment, instruments or materials are identified to adequately specify experimental conditions or reported results. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the equipment, instruments or materials identified are necessarily the best available for the purpose. C.F. was funded by Instituto de Salud Carlos III (PI20/00876) and Ministerio de Ciencia e Innovación (RTC-2017-6471-1; AEI/FEDER, UE), cofinanced by the European Regional Development Fund ‘A Way of Making Europe’ from the European Union, and Cabildo Insular de Tenerife (CGIEU0000219140). J.M.L.-S. was funded by Consejería de Educación-Gobierno de Canarias and Cabildo Insular de Tenerife (BOC 163, 24/08/2017). F.J.S. and M.M. was supported by the NIH (UM1 HG008898). C.X. was supported by the Intramural Research Program of the National Library of Medicine, NIH. K.H.M. was supported by the NIH/National Human Genome Research Institute (R01 1R01HG011274-01 and U01 1U01HG010971). H.L. was supported by the NIH (R01 HG010040 and U01 HG010961). C.E.M. thanks funding from the WorldQuant Foundation, NASA (NNX14AH50G), the National Institutes of Health (R01MH117406, R01CA249054, R01AI151059, P01CA214274) and the Leukemia and Lymphoma Society (LLS) (MCL7001-18, LLS 9238-16, LLS-MCL7001-18).

Author information

Authors and Affiliations



Conceptualization: J.W., N.D.O., A.F., K.H.M., S.E.L., M.T.W.E., H.L., C.-S.C., J.M.Z. and F.J.S. Data curation: J.W., N.D.O. and J.M. Formal analysis – benchmark: J.W., N.D.O., J.M. and J.M.Z. Formal analysis – assembly: H.C., A.S., H.L. and C.-S.C. Methodology: J.W., H.C., H.L., C.-S.C., J.M.Z. and F.J.S. Project administration: J.W., J.M.Z. and F.J.S. Resources: C.X. Software: J.W. and N.D.O. Supervision: C.-S.C., J.M.Z. and F.J.S. Validation: J.W., N.D.O., L.H., J.M., H.C., A.F., Y.-C.H., R.G., A.M.W., W.J.R., Z.M.K., J.F., Y.Z., A.P., M.M., C.X., B.Y., S.M.E.S., D.J., J.M.L.-S., A.M.-B., L.A.R.-R., C.F., G.N., U.S.E., S.E.C., J.L., H.L., C.-S.C., J.M.Z. and F.J.S. Visualization: J.W., N.D.O., H.C., H.L. and C.-S.C. Writing – original draft: J.W., L.H., C.-S.C., J.M.Z. and F.J.S. Writing – review and editing: J.W., N.D.O., D.E.M., J.L., C.E.M., S.E.L., M.T.W.E., C.-S.C., J.M.Z. and F.J.S.

Corresponding authors

Correspondence to Chen-Shan Chin, Justin M. Zook or Fritz J. Sedlazeck.

Ethics declarations

Competing interests

A.M.W. and W.J.R. are employees and shareholders of Pacific Biosciences. A.F., Y.-C.H, R.G., and C.-S.C. are employees and shareholders of DNAnexus. S.M.E.S. is an employee of Roche. J.L. is a former employee and shareholder of Bionano Genomics. S.E.L. was an employee of Invitae. F.J.S. has sponsored travel from Pacific Biosciences and Oxford Nanopore Technologies. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Adam Ameur, Christian Marshall and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figures 1–17, Notes 1–5 and Table 1.

Reporting Summary.

Supplementary Data 1

Additional characteristics of high-priority clinical genes.

Supplementary Data 2

Overlaps of the 5,038 genes on GRCh38 primary assembly between both HG002 GRCh38 v4.2.1 and HG002 hifiasm v0.11.

Supplementary Data 3

Benchmarking of the hifiasm v0.11 assembly-based variants called with dipcall against the GIAB v4.2.1 benchmark for HG002.

Supplementary Data 4

Benchmarking statistics against CMRG benchmark and evaluation callsets.

Supplementary Data 5

Manual curation results for evaluation and common errors in v0.02.03 small variant benchmark.

Supplementary Data 6

Primer designs and reaction conditions for Long-Range PCR and Sanger confirmation.

Supplementary Data 7

Genes excluded from the CMRG benchmarks, with likely reasons for exclusion annotated for GRCh38 in the last column.

Supplementary Data 8

Commands for BWA-GATK variant calling on normal GRCh38 reference.

Supplementary Data 9

Commands for BWA-GATK variant calling on v1 masked GRCh38 reference.

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Wagner, J., Olson, N.D., Harris, L. et al. Curated variation benchmarks for challenging medically relevant autosomal genes. Nat Biotechnol 40, 672–680 (2022).

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