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INTRODUCTION One of the central applications of the human reference genome has been to serve as a baseline for comparison in nearly all human genomic studies. Unfortunately, many difficult regions of the reference genome have remained unresolved for decades and are affected by collapsed duplications, missing sequences, and other issues. Relative to the current human reference genome, GRCh38, the Telomere-to-Telomere CHM13 (T2T-CHM13) genome closes all remaining gaps, adds nearly 200 million base pairs (Mbp) of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome for scientific inquiry. RATIONALE We demonstrate how the T2T-CHM13 reference genome universally improves read mapping and variant identification in a globally diverse cohort. This cohort includes all 3202 samples from the expanded 1000 Genomes Project (1KGP), sequenced with short reads, as well as 17 globally diverse samples sequenced with long reads. By applying state-of-the-art methods for calling single-nucleotide variants (SNVs) and structural variants (SVs), we document the strengths and limitations of T2T-CHM13 relative to its predecessors and highlight its promise for revealing new biological insights within technically challenging regions of the genome. RESULTS Across the 1KGP samples, we found more than 1 million additional high-quality variants genome-wide using T2T-CHM13 than with GRCh38. Within previously unresolved regions of the genome, we identified hundreds of thousands of variants per sample—a promising opportunity for evolutionary and biomedical discovery. T2T-CHM13 improves the Mendelian concordance rate among trios and eliminates tens of thousands of spurious SNVs per sample, including a reduction of false positives in 269 challenging, medically relevant genes by up to a factor of 12. These corrections are in large part due to improvements to 70 protein-coding genes in >9 Mbp of inaccurate sequence caused by falsely collapsed or duplicated regions in GRCh38. Using the T2T-CHM13 genome also yields a more comprehensive view of SVs genome-wide, with a greatly improved balance of insertions and deletions. Finally, by providing numerous resources for T2T-CHM13 (including 1KGP genotypes, accessibility masks, and prominent annotation databases), our work will facilitate the transition to T2T-CHM13 from the current reference genome. CONCLUSION The vast improvements in variant discovery across samples of diverse ancestries position T2T-CHM13 to succeed as the next prevailing reference for human genetics. T2T-CHM13 thus offers a model for the construction and study of high-quality reference genomes from globally diverse individuals, such as is now being pursued through collaboration with the Human Pangenome Reference Consortium. As a foundation, our work underscores the benefits of an accurate and complete reference genome for revealing diversity across human populations. Genomic features and resources available for T2T-CHM13. Comparisons to GRCh38 reveal broad improvements in SNVs, indels, and SVs discovered across diverse human populations by means of short-read (1KGP) and long-read sequencing (LRS). These improvements are due to resolution of complex genomic loci (nonsyntenic and previously unresolved), duplication errors, and discordant haplotypes, including those in medically relevant genes.
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This content will become publicly available on November 1, 2025
High-coverage nanopore sequencing of samples from the 1000 Genomes Project to build a comprehensive catalog of human genetic variation
Fewer than half of individuals with a suspected Mendelian or monogenic condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control data sets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project (1KGP) Oxford Nanopore Technologies Sequencing Consortium aims to generate LRS data from at least 800 of the 1KGP samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37× and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.
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- PAR ID:
- 10581408
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- Genome Research
- Volume:
- 34
- Issue:
- 11
- ISSN:
- 1088-9051
- Page Range / eLocation ID:
- 2061 to 2073
- Subject(s) / Keyword(s):
- human genomics bioinformatics structural variation long read sequencing
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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