Variant Calling and Benchmarking in an Era of Complete Human Genome Sequences

Overview

Journal Nat Rev Genet

Specialty Genetics

Date 2023 Apr 14

PMID 37059810

Authors

Nathan D Olson

Justin Wagner

Nathan Dwarshuis

Karen H Miga

Fritz J Sedlazeck

Marc Salit

Justin M Zook

Affiliations

Soon will be listed here.

Abstract

Genetic variant calling from DNA sequencing has enabled understanding of germline variation in hundreds of thousands of humans. Sequencing technologies and variant-calling methods have advanced rapidly, routinely providing reliable variant calls in most of the human genome. We describe how advances in long reads, deep learning, de novo assembly and pangenomes have expanded access to variant calls in increasingly challenging, repetitive genomic regions, including medically relevant regions, and how new benchmark sets and benchmarking methods illuminate their strengths and limitations. Finally, we explore the possible future of more complete characterization of human genome variation in light of the recent completion of a telomere-to-telomere human genome reference assembly and human pangenomes, and we consider the innovations needed to benchmark their newly accessible repetitive regions and complex variants.

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References

Olson N, Wagner J, McDaniel J, Stephens S, Westreich S, Prasanna A . PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions. Cell Genom. 2022; 2(5). PMC: 9205427. DOI: 10.1016/j.xgen.2022.100129. View

Pan B, Ren L, Onuchic V, Guan M, Kusko R, Bruinsma S . Assessing reproducibility of inherited variants detected with short-read whole genome sequencing. Genome Biol. 2022; 23(1):2. PMC: 8722114. DOI: 10.1186/s13059-021-02569-8. View

Foox J, Tighe S, Nicolet C, Zook J, Byrska-Bishop M, Clarke W . Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study. Nat Biotechnol. 2021; 39(9):1129-1140. PMC: 8985210. DOI: 10.1038/s41587-021-01049-5. View

Jain M, Koren S, Miga K, Quick J, Rand A, Sasani T . Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol. 2018; 36(4):338-345. PMC: 5889714. DOI: 10.1038/nbt.4060. View

Wenger A, Peluso P, Rowell W, Chang P, Hall R, Concepcion G . Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol. 2019; 37(10):1155-1162. PMC: 6776680. DOI: 10.1038/s41587-019-0217-9. View

Nurk S, Koren S, Rhie A, Rautiainen M, Bzikadze A, Mikheenko A . The complete sequence of a human genome. Science. 2022; 376(6588):44-53. PMC: 9186530. DOI: 10.1126/science.abj6987. View

Meacham F, Boffelli D, Dhahbi J, Martin D, Singer M, Pachter L . Identification and correction of systematic error in high-throughput sequence data. BMC Bioinformatics. 2011; 12:451. PMC: 3295828. DOI: 10.1186/1471-2105-12-451. View

Nurk S, Walenz B, Rhie A, Vollger M, Logsdon G, Grothe R . HiCanu: accurate assembly of segmental duplications, satellites, and allelic variants from high-fidelity long reads. Genome Res. 2020; 30(9):1291-1305. PMC: 7545148. DOI: 10.1101/gr.263566.120. View

Hannan A . Tandem repeats mediating genetic plasticity in health and disease. Nat Rev Genet. 2018; 19(5):286-298. DOI: 10.1038/nrg.2017.115. View

10.

Karczewski K, Francioli L, Tiao G, Cummings B, Alfoldi J, Wang Q . The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020; 581(7809):434-443. PMC: 7334197. DOI: 10.1038/s41586-020-2308-7. View

11.

Bakhtiari M, Shleizer-Burko S, Gymrek M, Bansal V, Bafna V . Targeted genotyping of variable number tandem repeats with adVNTR. Genome Res. 2018; 28(11):1709-1719. PMC: 6211647. DOI: 10.1101/gr.235119.118. View

12.

Vollger M, Guitart X, Dishuck P, Mercuri L, Harvey W, Gershman A . Segmental duplications and their variation in a complete human genome. Science. 2022; 376(6588):eabj6965. PMC: 8979283. DOI: 10.1126/science.abj6965. View

13.

Zhao X, Collins R, Lee W, Weber A, Jun Y, Zhu Q . Expectations and blind spots for structural variation detection from long-read assemblies and short-read genome sequencing technologies. Am J Hum Genet. 2021; 108(5):919-928. PMC: 8206509. DOI: 10.1016/j.ajhg.2021.03.014. View

14.

Lincoln S, Hambuch T, Zook J, Bristow S, Hatchell K, Truty R . One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation. Genet Med. 2021; 23(9):1673-1680. PMC: 8460443. DOI: 10.1038/s41436-021-01187-w. View

15.

Behera S, LeFaive J, Orchard P, Mahmoud M, Paulin L, Farek J . FixItFelix: improving genomic analysis by fixing reference errors. Genome Biol. 2023; 24(1):31. PMC: 9942314. DOI: 10.1186/s13059-023-02863-7. View

16.

Aganezov S, Yan S, Soto D, Kirsche M, Zarate S, Avdeyev P . A complete reference genome improves analysis of human genetic variation. Science. 2022; 376(6588):eabl3533. PMC: 9336181. DOI: 10.1126/science.abl3533. View

17.

Wagner J, Olson N, Harris L, McDaniel J, Cheng H, Fungtammasan A . Curated variation benchmarks for challenging medically relevant autosomal genes. Nat Biotechnol. 2022; 40(5):672-680. PMC: 9117392. DOI: 10.1038/s41587-021-01158-1. View

18.

Sudmant P, Rausch T, Gardner E, Handsaker R, Abyzov A, Huddleston J . An integrated map of structural variation in 2,504 human genomes. Nature. 2015; 526(7571):75-81. PMC: 4617611. DOI: 10.1038/nature15394. View

19.

Vollger M, Dishuck P, Sorensen M, Welch A, Dang V, Dougherty M . Long-read sequence and assembly of segmental duplications. Nat Methods. 2018; 16(1):88-94. PMC: 6382464. DOI: 10.1038/s41592-018-0236-3. View

20.

Cheng H, Concepcion G, Feng X, Zhang H, Li H . Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18(2):170-175. PMC: 7961889. DOI: 10.1038/s41592-020-01056-5. View