Characterization of subclonal variants in HG002 Genome in a Bottle reference material as a resource for benchmarking variant callers

Camille A Daniels¹, Adetola A Abdulkadir¹, Megan H Cleveland², Jennifer H McDaniel², David Jáspez³, Luis Alberto Rubio-Rodríguez³, Adrián Muñoz-Barrera³, José Miguel Lorenzo-Salazar³, Carlos Flores⁴, Byunggil Yoo⁵, Sayed Mohammad Ebrahim Sahraeian⁶, Yina Wang⁷, Massimiliano Rossi⁷, Arun Visvanath⁷, Lisa Murray⁷, Wei-Ting Chen⁷, Severine Catreux⁷, James Han⁷, Rami Mehio⁷, Gavin Parnaby⁷, Andrew Carroll⁸, Pi-Chuan Chang⁸, Kishwar Shafin⁸, Daniel Cook⁸, Alexey Kolesnikov⁸, Lucas Brambrink⁸, Mohammed Faizal Eeman Mootor⁹, Yash Patel⁹, Takafumi N Yamaguchi⁹, Paul C Boutros⁹, Karolina Sienkiewicz¹⁰, Jonathan Foox¹¹, Christopher E Mason¹², Bryan R Lajoie¹³, Carlos A Ruiz-Perez¹³, Semyon Kruglyak¹³, Justin M Zook¹⁴, Nathan D Olson¹⁵

Affiliations

¹ Medical Device Innovation Consortium (MDIC), 1655 N Ft. Myer Drive, Suite 250, Arlington, VA 22209, USA.
² Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA.
³ Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain.
⁴ Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain; Research Unit, Hospital Universitario Nuestra Señora de Candelaria, Instituto de Investigación Sanitaria de Canarias, Santa Cruz de Tenerife, Spain; CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain; Facultad de Ciencias de la Salud, Universidad Fernando de Pessoa Canarias, Las Palmas de Gran Canaria, Spain.
⁵ Genomic Medicine Center, Children's Mercy Kansas City, Kansas City, MO, USA.
⁶ Roche Sequencing Solutions, Santa Clara, CA 95050, USA.
⁷ Illumina, Inc., San Diego, CA, USA.
⁸ Google, Inc., Mountain View, CA, USA.
⁹ Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
¹⁰ Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA.
¹¹ Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.
¹² Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA; The WorldQuant Initiative for Quantitative Prediction, Weill Cornell Medicine, New York, NY, USA.
¹³ Element Biosciences, San Diego, CA, USA.
¹⁴ Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA. Electronic address: justin.zook@nist.gov.
¹⁵ Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA. Electronic address: nolson@nist.gov.

PMID: 41421359
DOI: 10.1016/j.xgen.2025.101104

Free article

Characterization of subclonal variants in HG002 Genome in a Bottle reference material as a resource for benchmarking variant callers

Camille A Daniels et al. Cell Genom. 2025.

Free article

. 2025 Dec 19:101104.

doi: 10.1016/j.xgen.2025.101104. Online ahead of print.

Authors

Affiliations

¹ Medical Device Innovation Consortium (MDIC), 1655 N Ft. Myer Drive, Suite 250, Arlington, VA 22209, USA.
² Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA.
³ Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain.
⁴ Genomics Division, Instituto Tecnológico y de Energías Renovables (ITER), Santa Cruz de Tenerife, Spain; Research Unit, Hospital Universitario Nuestra Señora de Candelaria, Instituto de Investigación Sanitaria de Canarias, Santa Cruz de Tenerife, Spain; CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain; Facultad de Ciencias de la Salud, Universidad Fernando de Pessoa Canarias, Las Palmas de Gran Canaria, Spain.
⁵ Genomic Medicine Center, Children's Mercy Kansas City, Kansas City, MO, USA.
⁶ Roche Sequencing Solutions, Santa Clara, CA 95050, USA.
⁷ Illumina, Inc., San Diego, CA, USA.
⁸ Google, Inc., Mountain View, CA, USA.
⁹ Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA, USA.
¹⁰ Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA.
¹¹ Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.
¹² Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA; The WorldQuant Initiative for Quantitative Prediction, Weill Cornell Medicine, New York, NY, USA.
¹³ Element Biosciences, San Diego, CA, USA.
¹⁴ Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA. Electronic address: justin.zook@nist.gov.
¹⁵ Material Measurement Laboratory, National Institute of Standards and Technology (NIST), 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA. Electronic address: nolson@nist.gov.

PMID: 41421359
DOI: 10.1016/j.xgen.2025.101104

Abstract

We developed a benchmark set of subclonal variants in the Genome in a Bottle (GIAB) Consortium HG002 reference material (RM) DNA for evaluating lower-frequency variant callsets. We used a somatic variant caller with high-coverage (300×) whole-genome sequencing data from the GIAB Ashkenazi Jewish trio to identify potential subclonal variants in the HG002 RM DNA. Using orthogonal sequencing data and manual curation, we defined a benchmark set with 85 high-confidence subclonal single-nucleotide variants (SNVs) (allele frequency [AF] > 5%) and a benchmark region covering 2.45 Gbp of the autosomes. External validation supported that it can be used to reliably identify both false negatives and false positives for a variety of sequencing technologies and variant callers. By adding our characterization of mosaic SNVs in this widely used cell line, we have expanded the scope of bioinformatic and sequencing applications for which the HG002 GIAB RM can be used to include benchmarking subclonal SNVs.

Keywords: Genome In A Bottle; SNV; genome sequencing; mosaic variant; reference material; somatic mosaicism; somatic variant; variant benchmarking; variant calling.

Published by Elsevier Inc.

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Conflict of interest statement

Declaration of interests C.E.M. is a co-founder of Onegevity. Y.W., M.R., A.V., L.M., W.-T.C., S.C., J.H., R.M., and G.P. are Illumina employees and equity owners. A.C., P.-C.C., K. Shafin, D.C., A.K., and L.B. are employees of Google LLC and receive equity compensation. P.C.B. sits on the scientific advisory boards of Intersect Diagnostics, Inc., and BioSymetrics, Inc., and previously sat on that of Sage Bionetworks.

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Characterization of subclonal variants in HG002 Genome in a Bottle reference material as a resource for benchmarking variant callers

Affiliations

Characterization of subclonal variants in HG002 Genome in a Bottle reference material as a resource for benchmarking variant callers

Authors

Affiliations

Abstract

Conflict of interest statement

LinkOut - more resources

Full Text Sources

Research Materials