TandemTools: mapping long reads and assessing/improving assembly quality in extra-long tandem repeats

Mikheenko, Alla; Bzikadze, Andrey V.; Gurevich, Alexey; Miga, Karen H.; Pevzner, Pavel A.

doi:10.1093/bioinformatics/btaa440

Cited by 55 publications

(64 citation statements)

References 54 publications

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“…7c–j , Supplementary Table 4 ). Mapped long-read coverage across the DXZ1 array shows uniform depth of coverage and high accuracy, as measured by TandemQUAST 41 (Fig. 2 e, f , Extended Data Figs.…”

Section: A Finished Human X Chromosomementioning

confidence: 91%

Telomere-to-telomere assembly of a complete human X chromosome

Miga

Koren

Rhie

et al. 2020

Nature

643

660

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After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes. Complete, telomere-to-telomere reference genome assemblies are necessary to ensure that all genomic variants are discovered and studied. At present, unresolved areas of the human genome are defined by multi-megabase satellite arrays in the pericentromeric regions and the ribosomal DNA arrays on acrocentric short arms, as well as regions enriched in segmental duplications that are greater than hundreds of kilobases in length and that exhibit sequence identity of more than 98% between paralogues. Owing to their absence from the reference, these repeat-rich sequences are often excluded from genetics and genomics studies, which limits the scope of association and functional analyses 4,5. Unresolved repeat sequences also result in unintended consequences; for example, paralogous sequence variants incorrectly being called as allelic variants 6 , and the contamination of bacterial gene databases 7. Completion of the entire human genome is expected to contribute to our understanding of chromosome function 8 , human disease 9 and genomic variation, which will improve technologies in biomedicine that use short-read mapping to a reference genome (for example, RNA sequencing (RNA-seq) 10 , chromatin immunoprecipitation followed by sequencing (ChIP-seq) 11 and assay for transposase-accessible chromatin using sequencing (ATAC-seq) 12). The fundamental challenge of reconstructing a genome from many comparatively short sequencing reads-a process known as genome assembly-is distinguishing the repeated sequences from one another 13. Resolving such repeats relies on sequencing reads that are long enough to span the entire repeat or accurate enough to distinguish each repeat copy on the basis of...

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Section: A Finished Human X Chromosomementioning

confidence: 91%

Telomere-to-telomere assembly of a complete human X chromosome

Miga

Koren

Rhie

et al. 2020

Nature

643

660

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“…We assessed the structure of the CHM13 and NHP centromeric HOR arrays by applying TandemMapper and TandemQUAST 52 ( https://github.com/ablab/TandemTools ; version from 20 March 2020), which can detect large structural assembly errors in repeat arrays. For the CHM13 centromere, we first aligned ONT reads longer than 50 kb to the CHM13 assembly containing the contiguous chromosome 8 with Winnowmap 43 (v.1.0) and extracted reads aligning to the centromeric HOR array (chr8:44243868–46323885).…”

Section: Methodsmentioning

confidence: 99%

The structure, function and evolution of a complete human chromosome 8

Logsdon

Vollger

Hsieh

et al. 2021

Nature

Self Cite

273

278

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The complete assembly of each human chromosome is essential for understanding human biology and evolution1,2. Here we use complementary long-read sequencing technologies to complete the linear assembly of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the β-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.

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“…The tools for the reconciliation of long tandem arrays, and their comparisons, are currently being developed [ 86 ]. Satellite arrays inside the existing assemblies/references can be curated, e.g., with TandemTools, a novel tool for the polishing and quality assessment of extra-long tandem repeats (ETRs) [ 87 ]. Specific long-read mappers can be used to align reads to highly repetitive reference sequences [ 88 ], while accounting for the allele bias (so that non-reference allele within a repeat does not penalize the alignment) [ 89 ].…”

Section: Long Readsmentioning

confidence: 99%

Probably Correct: Rescuing Repeats with Short and Long Reads

Čechová

2020

Genes

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Ever since the introduction of high-throughput sequencing following the human genome project, assembling short reads into a reference of sufficient quality posed a significant problem as a large portion of the human genome—estimated 50–69%—is repetitive. As a result, a sizable proportion of sequencing reads is multi-mapping, i.e., without a unique placement in the genome. The two key parameters for whether or not a read is multi-mapping are the read length and genome complexity. Long reads are now able to span difficult, heterochromatic regions, including full centromeres, and characterize chromosomes from “telomere to telomere”. Moreover, identical reads or repeat arrays can be differentiated based on their epigenetic marks, such as methylation patterns, aiding in the assembly process. This is despite the fact that long reads still contain a modest percentage of sequencing errors, disorienting the aligners and assemblers both in accuracy and speed. Here, I review the proposed and implemented solutions to the repeat resolution and the multi-mapping read problem, as well as the downstream consequences of reference choice, repeat masking, and proper representation of sex chromosomes. I also consider the forthcoming challenges and solutions with regards to long reads, where we expect the shift from the problem of repeat localization within a single individual to the problem of repeat positioning within pangenomes.

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TandemTools: mapping long reads and assessing/improving assembly quality in extra-long tandem repeats

Cited by 55 publications

References 54 publications

Telomere-to-telomere assembly of a complete human X chromosome

Telomere-to-telomere assembly of a complete human X chromosome

The structure, function and evolution of a complete human chromosome 8

Probably Correct: Rescuing Repeats with Short and Long Reads

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