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

2019 ◽  
Author(s):  
Karen H. Miga ◽  
Sergey Koren ◽  
Arang Rhie ◽  
Mitchell R. Vollger ◽  
Ariel Gershman ◽  
...  
Keyword(s):  
Human Genome ◽  
X Chromosome ◽  
Satellite Dna ◽  
Tandem Repeats ◽  
Reference Genome ◽  
De Novo ◽  
Hydatidiform Mole ◽  
Segmental Duplications ◽  
High Coverage ◽  
Current Reference

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first 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 ∼2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

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

Nature ◽  
2020 ◽  
Vol 585 (7823) ◽  
pp. 79-84 ◽  
Author(s):  
Karen H. Miga ◽  
Sergey Koren ◽  
Arang Rhie ◽  
Mitchell R. Vollger ◽  
Ariel Gershman ◽  
...  
Keyword(s):  
Human Genome ◽  
X Chromosome ◽  
Tandem Repeats ◽  
Reference Genome ◽  
Hydatidiform Mole ◽  
Gene Families ◽  
High Coverage ◽  
Single Chromosome ◽  
Human Reference Genome ◽  
Pseudoautosomal Regions

AbstractAfter 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 persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, 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 chromosome3, 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.

scholarly journals Recovery of non-reference sequences missing from the human reference genome

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ran Li ◽  
Xiaomeng Tian ◽  
Peng Yang ◽  
Yingzhi Fan ◽  
Ming Li ◽  
...  
Keyword(s):  
Human Genome ◽  
Tandem Repeats ◽  
Reference Genome ◽  
De Novo ◽  
Precise Location ◽  
Protein Coding ◽  
Human Reference Genome ◽  
Mhc Haplotype ◽  
Reference Sequences ◽  
Flanking Regions

Abstract Background The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Results Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6113 NRS adding up to 12.8 Mb. Besides 1571 insertions, we detected 3041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Conclusions Our study detected a large number of NRS including many alternate alleles which are previously uncharacterized. We suggested that the origin of alternate alleles was associated with tandem repeats. Our results enriched the spectrum of genetic variations in human genome.

scholarly journals Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

2019 ◽  
Author(s):  
Mitchell R. Vollger ◽  
Glennis A. Logsdon ◽  
Peter A. Audano ◽  
Arvis Sulovari ◽  
David Porubsky ◽  
...  
Keyword(s):  
Human Genome ◽  
Single Molecule ◽  
Tandem Repeats ◽  
De Novo ◽  
Sequence Data ◽  
Gene Annotation ◽  
Hydatidiform Mole ◽  
High Fidelity ◽  
Human Genomes ◽  
Long Read

AbstractThe sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective stand-alone technology for de novo assembly of human genomes.

scholarly journals Recovery of non-reference sequences missing from the human reference genome

2019 ◽  
Author(s):  
Ran Li ◽  
Xiaomeng Tian ◽  
Peng Yang ◽  
Yingzhi Fan ◽  
Ming Li ◽  
...  
Keyword(s):  
Human Genome ◽  
Tandem Repeats ◽  
Reference Genome ◽  
De Novo ◽  
Precise Location ◽  
Protein Coding ◽  
Human Reference Genome ◽  
Mhc Haplotype ◽  
Reference Sequences ◽  
Flanking Regions

Abstract Background The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Results Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6,113 NRS adding up to 12.8 Mb. Besides 1,571 insertions, we detected 3,041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1,143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Conclusions Our study detected a large number of NRS including many alternate alleles which are previously uncharacterized. We suggested that the origin of alternate alleles was associated with tandem repeats. Our results enriched the spectrum of genetic variations in human genome.

scholarly journals The X Chromosome from Telomere to Telomere: Key Achievements and Future Opportunities

2021 ◽  
Vol 10 ◽  
Author(s):  
Edith Heard ◽  
Alexander D Johnson ◽  
Jan O Korbel ◽  
Charles Lee ◽  
Michael P Snyder ◽  
...  
Keyword(s):  
Human Genome ◽  
X Chromosome ◽  
Tandem Repeats ◽  
Hydatidiform Mole ◽  
Dark Side ◽  
Dna Array ◽  
Complete Hydatidiform Mole ◽  
Long Read ◽  
Methylation Patterns ◽  
Large Repeat

While the human genome represents the most accurate vertebrate reference assembly to date, it still contains numerous gaps, including centromeric and other large repeat-containing regions – often termed the “dark side” of the genome – many of which are of fundamental biological importance. Miga et al. present the first gapless assembly of the human X chromosome, with the help of ultra-long-read nanopore reads generated for the haploid complete hydatidiform mole (CHM13) genome. They reconstruct the ~3.1 megabase centromeric satellite DNA array and map DNA methylation patterns across complex tandem repeats and satellite arrays. This Telomere-to-Telomere assembly provides a superior human X chromosome reference enabling future sex-determination and X-linked disease research, and provides a path towards finishing the entire human genome sequence.

scholarly journals Recovery of non-reference sequences missing from the human reference genome

2019 ◽  
Author(s):  
Ran Li ◽  
Xiaomeng Tian ◽  
Peng Yang ◽  
Yingzhi Fan ◽  
Ming Li ◽  
...  
Keyword(s):  
Human Genome ◽  
Tandem Repeats ◽  
Reference Genome ◽  
De Novo ◽  
Precise Location ◽  
Protein Coding ◽  
Human Reference Genome ◽  
Mhc Haplotype ◽  
Reference Sequences ◽  
Flanking Regions

Abstract Background The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Results Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6,113 NRS adding up to 12.8 Mb. Besides 1,571 insertions, we detected 3,041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1,143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Conclusions Our study detected a large number of NRS including many alternate alleles which are previously uncharacterized. We suggested that the origin of alternate alleles was associated with tandem repeats. Our results enriched the spectrum of genetic variations in human genome.

scholarly journals Centromeric Satellite DNAs: Hidden Sequence Variation in the Human Population

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 352 ◽  
Author(s):  
Karen H. Miga
Keyword(s):  
Human Genome ◽  
Satellite Dna ◽  
Human Population ◽  
Sequence Variation ◽  
Tandem Repeats ◽  
Association Studies ◽  
Unmet Need ◽  
Satellite Dnas ◽  
Dna Variation ◽  
Medical Genomics

The central goal of medical genomics is to understand the inherited basis of sequence variation that underlies human physiology, evolution, and disease. Functional association studies currently ignore millions of bases that span each centromeric region and acrocentric short arm. These regions are enriched in long arrays of tandem repeats, or satellite DNAs, that are known to vary extensively in copy number and repeat structure in the human population. Satellite sequence variation in the human genome is often so large that it is detected cytogenetically, yet due to the lack of a reference assembly and informatics tools to measure this variability, contemporary high-resolution disease association studies are unable to detect causal variants in these regions. Nevertheless, recently uncovered associations between satellite DNA variation and human disease support that these regions present a substantial and biologically important fraction of human sequence variation. Therefore, there is a pressing and unmet need to detect and incorporate this uncharacterized sequence variation into broad studies of human evolution and medical genomics. Here I discuss the current knowledge of satellite DNA variation in the human genome, focusing on centromeric satellites and their potential implications for disease.

scholarly journals Rapid low-cost assembly of the Drosophila melanogaster reference genome using low-coverage, long-read sequencing

2018 ◽  
Author(s):  
Edwin A. Solares ◽  
Mahul Chakraborty ◽  
Danny E. Miller ◽  
Shannon Kalsow ◽  
Kate Hall ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Genetic Variation ◽  
Large Scale ◽  
Reference Genome ◽  
De Novo ◽  
Low Cost ◽  
Nucleotide Polymorphisms ◽  
Structural Variants ◽  
High Coverage ◽  
Reference Assembly

ABSTRACTAccurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from large-scale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using high-coverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hours. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD).

scholarly journals Illumina TruSeq synthetic long-reads empowerde novoassembly and resolve complex, highly repetitive transposable elements

2014 ◽  
Author(s):  
Rajiv C McCoy ◽  
Ryan W Taylor ◽  
Timothy A Blauwkamp ◽  
Joanna L Kelley ◽  
Michael Kertesz ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Reference Genome ◽  
De Novo ◽  
Model Organism ◽  
Genomic Analysis ◽  
High Sequence Identity ◽  
Current Reference ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Whole Genomes

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including thede novoassembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or present in complex genomic arrangements. While TEs strongly affect genome function and evolution, most currentde novoassembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly parallel library preparation and local assembly of short read data and achieve lengths of 1.5-18.5 Kbp with an extremely low error rate (∼0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organismDrosophila melanogaster(reference genome strainy;cn,bw,sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 of annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long- reads, and likely other methods that generate long reads, offer a powerful approach to improvede novoassemblies of whole genomes.

scholarly journals Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies

2021 ◽  
Author(s):  
Arang Rhie ◽  
Ann Mc Cartney ◽  
Kishwar Shafin ◽  
Michael Alonge ◽  
Andrey Bzikadze ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Tandem Repeats ◽  
Hydatidiform Mole ◽  
Segmental Duplications ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Human Genome Assembly ◽  
Long Read ◽  
Genome Assemblies ◽  
Oxford Nanopore Technologies

Abstract Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first Telomere-to-Telomere (T2T) human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Though derived from highly accurate sequencing, evaluation revealed that the initial T2T draft assembly had evidence of small errors and structural misassemblies. To correct these errors, we designed a novel repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly QV to 73.9. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both PacBio HiFi and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies

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