Human Genome Assembly Research Articles

Complete analysis of G-quadruplex forming sequences in the gapless assembly of human chromosome Y

Recent advancements have finally delivered a complete human genome assembly, including the elusive Y chromosome. This accomplishment closes a significant knowledge gap. Prior efforts were hampered by challenges in sequencing repetitive DNA structures such as direct and inverted repeats. We used the G4Hunter algorithm to analyze the presence of G-quadruplex forming sequences (G4s) within the current human reference genome (GRCh38) and the new telomere-to-telomere (T2T) Y chromosome assemblies. This analysis served a dual purpose: identifying the location of potential G4s within the genomes and exploring their association with functionally annotated sequences. Compared to GRCh38, the T2T assembly exhibited a significantly higher prevalence of G-quadruplex forming sequences. Notably, these repeats were abundantly located around precursor RNA, exons, genes, and within protein binding sites. This remarkable co-occurrence of G4-forming sequences with these critical regulatory regions suggests their role in fundamental DNA regulation processes. Our findings indicate that the current human reference genome significantly underestimated the number of G4s, potentially overlooking their functional importance.

Variant graph craft (VGC): a comprehensive tool for analyzing genetic variation and identifying disease-causing variants

BackgroundThe variant call format (VCF) file is a structured and comprehensive text file crucial for researchers and clinicians in interpreting and understanding genomic variation data. It contains essential information about variant positions in the genome, along with alleles, genotype calls, and quality scores. Analyzing and visualizing these files, however, poses significant challenges due to the need for diverse resources and robust features for in-depth exploration.ResultsTo address these challenges, we introduce variant graph craft (VGC), a VCF file visualization and analysis tool. VGC offers a wide range of features for exploring genetic variations, including extraction of variant data, intuitive visualization, and graphical representation of samples with genotype information. VGC is designed primarily for the analysis of patient cohorts, but it can also be adapted for use with individual probands or families. It integrates seamlessly with external resources, providing insights into gene function and variant frequencies in sample data. VGC includes gene function and pathway information from Molecular Signatures Database (MSigDB) for GO terms, KEGG, Biocarta, Pathway Interaction Database, and Reactome. Additionally, it dynamically links to gnomAD for variant information and incorporates ClinVar data for pathogenic variant information. VGC supports the Human Genome Assembly Hg37 and Hg38, ensuring compatibility with a wide range of data sets, and accommodates various approaches to exploring genetic variation data. It can be tailored to specific user needs with optional phenotype input data.ConclusionsIn summary, VGC provides a comprehensive set of features tailored to researchers working with genomic variation data. Its intuitive interface, rapid filtering capabilities, and the flexibility to perform queries using custom groups make it an effective tool in identifying variants potentially associated with diseases. VGC operates locally, ensuring data security and privacy by eliminating the need for cloud-based VCF uploads, making it a secure and user-friendly tool. It is freely available at https://github.com/alperuzun/VGC.

Genetic complexity of killer-cell immunoglobulin-like receptor genes in human pangenome assemblies.

The killer-cell immunoglobulin-like receptor (KIR) gene complex, a highly polymorphic region of the human genome that encodes proteins involved in immune responses, poses strong challenges in genotyping owing to its remarkable genetic diversity and structural intricacy. Accurate analysis of KIR alleles, including their structural variations, is crucial for understanding their roles in various immune responses. Leveraging the high-quality genome assemblies from the Human Pangenome Reference Consortium (HPRC), we present a novel bioinformatic tool, the structural KIR annoTator (SKIRT), to investigate gene diversity and facilitate precise KIR allele analysis. In 47 HPRC-phased assemblies, SKIRT identifies a recurrent novel KIR2DS4/3DL1 fusion gene in the paternal haplotype of HG02630 and maternal haplotype of NA19240. Additionally, SKIRT accurately identifies eight structural variants and 15 novel nonsynonymous alleles, all of which are independently validated using short-read data or quantitative polymerase chain reaction. Our study has discovered a total of 570 novel alleles, among which eight haplotypes harbor at least one KIR gene duplication, six haplotypes have lost at least one framework gene, and 75 out of 94 haplotypes (79.8%) carry at least five novel alleles, thus confirming KIR genetic diversity. These findings are pivotal in providing insights into KIR gene diversity and serve as a solid foundation for understanding the functional consequences of KIR structural variations. High-resolution genome assemblies offer unprecedented opportunities to explore polymorphic regions that are challenging to investigate using short-read sequencing methods. The SKIRT pipeline emerges as a highly efficient tool, enabling the comprehensive detection of the complete spectrum of KIR alleles within human genome assemblies.

IGLoo: Profiling the Immunoglobulin Heavy chain locus in Lymphoblastoid Cell Lines with PacBio High-Fidelity Sequencing reads.

New high-quality human genome assemblies derived from lymphoblastoid cell lines (LCLs) provide reference genomes and pangenomes for genomics studies. However, the characteristics of LCLs pose technical challenges to profiling immunoglobulin (IG) genes. IG loci in LCLs contain a mixture of germline and somatically recombined haplotypes, making them difficult to genotype or assemble accurately. To address these challenges, we introduce IGLoo, a software tool that implements novel methods for analyzing sequence data and genome assemblies derived from LCLs. IGLoo characterizes somatic V(D)J recombination events in the sequence data and identifies the breakpoints and missing IG genes in the LCL-based assemblies. Furthermore, IGLoo implements a novel reassembly framework to improve germline assembly quality by integrating information about somatic events and population structural variantions in the IG loci. We applied IGLoo to study the assemblies from the Human Pangenome Reference Consortium, providing new insights into the mechanisms, gene usage, and patterns of V(D)J recombination, causes of assembly fragmentation in the IG heavy chain (IGH) locus, and improved representation of the IGH assemblies.

Novel Cascade Alpha Satellite HORs in Orangutan Chromosome 13 Assembly: Discovery of the 59mer HOR-The largest Unit in Primates-And the Missing Triplet 45/27/18 HOR in Human T2T-CHM13v2.0 Assembly.

From the recent genome assembly NHGRI_mPonAbe1-v2.0_NCBI (GCF_028885655.2) of orangutan chromosome 13, we computed the precise alpha satellite higher-order repeat (HOR) structure using the novel high-precision GRM2023 algorithm with Global Repeat Map (GRM) and Monomer Distance (MD) diagrams. This study rigorously identified alpha satellite HORs in the centromere of orangutan chromosome 13, discovering a novel 59mer HOR-the longest HOR unit identified in any primate to date. Additionally, it revealed the first intertwined sequence of three HORs, 18mer/27mer/45mer HORs, with a common aligned "backbone" across all HOR copies. The major 7mer HOR exhibits a Willard's-type canonical copy, although some segments of the array display significant irregularities. In contrast, the 14mer HOR forms a regular Willard's-type HOR array. Surprisingly, the GRM2023 high-precision analysis of chromosome 13 of human genome assembly T2T-CHM13v2.0 reveals the presence of only a 7mer HOR, despite both the orangutan and human genome assemblies being derived from whole genome shotgun sequences.

Simultaneous de novo calling and phasing of genetic variants at chromosome-scale using NanoStrand-seq

The successful accomplishment of the first telomere-to-telomere human genome assembly, T2T-CHM13, marked a milestone in achieving completeness of the human reference genome. The upcoming era of genome study will focus on fully phased diploid genome assembly, with an emphasis on genetic differences between individual haplotypes. Most existing sequencing approaches only achieved localized haplotype phasing and relied on additional pedigree information for further whole-chromosome scale phasing. The short-read-based Strand-seq method is able to directly phase single nucleotide polymorphisms (SNPs) at whole-chromosome scale but falls short when it comes to phasing structural variations (SVs). To shed light on this issue, we developed a Nanopore sequencing platform-based Strand-seq approach, which we named NanoStrand-seq. This method allowed for de novo SNP calling with high precision (99.52%) and acheived a superior phasing accuracy (0.02% Hamming error rate) at whole-chromosome scale, a level of performance comparable to Strand-seq for haplotype phasing of the GM12878 genome. Importantly, we demonstrated that NanoStrand-seq can efficiently resolve the MHC locus, a highly polymorphic genomic region. Moreover, NanoStrand-seq enabled independent direct calling and phasing of deletions and insertions at whole-chromosome level; when applied to long genomic regions of SNP homozygosity, it outperformed the strategy that combined Strand-seq with bulk long-read sequencing. Finally, we showed that, like Strand-seq, NanoStrand-seq was also applicable to primary cultured cells. Together, here we provided a novel methodology that enabled interrogation of a full spectrum of haplotype-resolved SNPs and SVs at whole-chromosome scale, with broad applications for species with diploid or even potentially polypoid genomes.

A comprehensive tandem repeat catalog of the human genome.

With the increasing availability of long-read sequencing data, high-quality human genome assemblies, and software for fully characterizing tandem repeats, genome-wide genotyping of tandem repeat loci on a population scale becomes more feasible. Such efforts not only expand our knowledge of the tandem repeat landscape in the human genome but also enhance our ability to differentiate pathogenic tandem repeat mutations from benign polymorphisms. To this end, we analyzed 272 genomes assembled using datasets from three public initiatives that employed different long-read sequencing technologies. Here, we report a catalog of over 18 million tandem repeat loci, many of which were previously unannotated. Some of these loci are highly polymorphic, and many of them reside within coding sequences.

Structural polymorphism and diversity of human segmental duplications.

Segmental duplications (SDs) contribute significantly to human disease, evolution, and diversity yet have been difficult to resolve at the sequence level. We present a population genetics survey of SDs by analyzing 170 human genome assemblies where the majority of SDs are fully resolved using long-read sequence assembly. Excluding the acrocentric short arms, we identify 173.2 Mbp of duplicated sequence (47.4 Mbp not present in the telomere-to-telomere reference) distinguishing fixed from structurally polymorphic events. We find that intrachromosomal SDs are among the most variable with rare events mapping near their progenitor sequences. African genomes harbor significantly more intrachromosomal SDs and are more likely to have recently duplicated gene families with higher copy number when compared to non-African samples. A comparison to a resource of 563 million full-length Iso-Seq reads identifies 201 novel, potentially protein-coding genes corresponding to these copy number polymorphic SDs.

Full-resolution HLA and KIR gene annotations for human genome assemblies.

The human leukocyte antigen (HLA) genes and the killer cell immunoglobulin-like receptor (KIR) genes are critical to immune responses and are associated with many immune-related diseases. Located in highly polymorphic regions, it is difficult to study them with traditional short-read alignment-based methods. Although modern long-read assemblers can often assemble these genes, using existing tools to annotate HLA and KIR genes in these assemblies remains a nontrivial task. Here, we describe Immuannot, a new computation tool to annotate the gene structures of HLA and KIR genes and to type the allele of each gene. Applying Immuannot to 56 regional and 212 whole-genome assemblies from previous studies, we annotate 9931 HLA and KIR genes and found that almost half of these genes, 4068, have novel sequences compared with the current Immuno Polymorphism Database (IPD). These novel gene sequences are represented by 2664 distinct alleles, some of which contained nonsynonymous variations, resulting in 92 novel protein sequences. We demonstrate the complex haplotype structures at the two loci and report the linkage between HLA/KIR haplotypes and gene alleles. We anticipate that Immuannot will speed up the discovery of new HLA/KIR alleles and enable the association of HLA/KIR haplotype structures with clinical outcomes in the future.

Characterization of the genetic variation and evolutionary divergence of the CLEC18 family.

The C-type lectin family 18 (CLEC18) with lipid and glycan binding capabilities is important to metabolic regulation and innate immune responses against viral infection. However, human CLEC18 comprises three paralogous genes with highly similar sequences, making it challenging to distinguish genetic variations, expression patterns, and biological functions of individual CLEC18 paralogs. Additionally, the evolutionary relationship between human CLEC18 and its counterparts in other species remains unclear. To identify the sequence variation and evolutionary divergence of human CLEC18 paralogs, we conducted a comprehensive analysis using various resources, including human and non-human primate reference genome assemblies, human pangenome assemblies, and long-read-based whole-genome and -transcriptomesequencing datasets. We uncovered paralogous sequence variants (PSVs) and polymorphic variants (PVs) of human CLEC18 proteins, and identified distinct signatures specific to each CLEC18 paralog. Furthermore, we unveiled a novel segmental duplication for human CLEC18A gene. By comparing CLEC18 across human and non-human primates, our research showed that the CLEC18 paralogy probably occurred in the common ancestor of human and closely related non-human primates, and the lipid-binding CAP/SCP/TAPS domain of CLEC18 is more diverse than its glycan-binding CTLD. Moreover, we found that certain amino acids alterations at variant positions are exclusive to human CLEC18 paralogs. Our findings offer a comprehensive profiling of the intricate variations and evolutionary characteristics of human CLEC18.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

LiftoffTools: a toolkit for comparing gene annotations mapped between genome assemblies.

In 2020 we published Liftoff, which was the first standalone tool specifically designed for transferring gene annotations between genome assemblies of the same or closely related species. While the gene content is expected to be very similar in closely related genomes, the differences may be biologically consequential, and a computational method to extract all gene-related differences should prove useful in the analysis of such genomes. Here we present LiftoffTools, a toolkit to automate the detection and analysis of gene sequence variants, synteny, and gene copy number changes. We provide a description of the toolkit and an example of its use comparing genes mapped between two human genome assemblies.

The Diverse Evolutionary Histories of Domesticated Metaviral Capsid Genes in Mammals.

Selfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae, giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals.

Co-linear chaining on pangenome graphs

Pangenome reference graphs are useful in genomics because they compactly represent the genetic diversity within a species, a capability that linear references lack. However, efficiently aligning sequences to these graphs with complex topology and cycles can be challenging. The seed-chain-extend based alignment algorithms use co-linear chaining as a standard technique to identify a good cluster of exact seed matches that can be combined to form an alignment. Recent works show how the co-linear chaining problem can be efficiently solved for acyclic pangenome graphs by exploiting their small width and how incorporating gap cost in the scoring function improves alignment accuracy. However, it remains open on how to effectively generalize these techniques for general pangenome graphs which contain cycles. Here we present the first practical formulation and an exact algorithm for co-linear chaining on cyclic pangenome graphs. We rigorously prove the correctness and computational complexity of the proposed algorithm. We evaluate the empirical performance of our algorithm by aligning simulated long reads from the human genome to a cyclic pangenome graph constructed from 95 publicly available haplotype-resolved human genome assemblies. While the existing heuristic-based algorithms are faster, the proposed algorithm provides a significant advantage in terms of accuracy. Implementation (https://github.com/at-cg/PanAligner).

Evolutionary insights from profiling LINE-1 activity at allelic resolution in a single human genome

Transposable elements have created the majority of the sequence in many genomes. In mammals, LINE-1 retrotransposons have been expanding for more than 100 million years as distinct, consecutive lineages; however, the drivers of this recurrent lineage emergence and disappearance are unknown. Most human genome assemblies provide a record of this ancient evolution, but fail to resolve ongoing LINE-1 retrotranspositions. Utilizing the human CHM1 long-read-based haploid assembly, we identified and cloned all full-length, intact LINE-1s, and found 29 LINE-1s with measurable in vitro retrotransposition activity. Among individuals, these LINE-1s varied in their presence, their allelic sequences, and their activity. We found that recently retrotransposed LINE-1s tend to be active in vitro and polymorphic in the population relative to more ancient LINE-1s. However, some rare allelic forms of old LINE-1s retain activity, suggesting older lineages can persist longer than expected. Finally, in LINE-1s with in vitro activity and in vivo fitness, we identified mutations that may have increased replication in ancient genomes and may prove promising candidates for mechanistic investigations of the drivers of LINE-1 evolution and which LINE-1 sequences contribute to human disease.

ELOA3: A primate-specific RNA polymerase II elongation factor encoded by a tandem repeat gene cluster.

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.

Tandem NBPF 3mer HORs (Olduvai triplets) in Neanderthal and two novel HOR tandem arrays in human chromosome 1 T2T-CHM13 assembly

It is known that the ~ 1.6 kb Neuroblastoma BreakPoint Family (NBPF) repeats are human specific and contributing to cognitive capabilities, with increasing frequency in higher order repeat 3mer HORs (Olduvai triplets). From chimpanzee to modern human there is a discontinuous jump from 0 to ~ 50 tandemly organized 3mer HORs. Here we investigate the structure of NBPF 3mer HORs in the Neanderthal genome assembly of Pääbo et al., comparing it to the results obtained for human hg38.p14 chromosome 1. Our findings reveal corresponding NBPF 3mer HOR arrays in Neanderthals with slightly different monomer structures and numbers of HOR copies compared to humans. Additionally, we compute the NBPF 3mer HOR pattern for the complete telomere-to-telomere human genome assembly (T2T-CHM13) by Miga et al., identifying two novel tandem arrays of NBPF 3mer HOR repeats with 5 and 9 NBPF 3mer HOR copies. We hypothesize that these arrays correspond to novel NBPF genes (here referred to as NBPFA1 and NBPFA2). Further improving the quality of the Neanderthal genome using T2T-CHM13 as a reference would be of great interest in determining the presence of such distant novel NBPF genes in the Neanderthal genome and enhancing our understanding of human evolution.

Efficient construction of the BWT for repetitive text using string compression

We present a new semi-external algorithm that builds the Burrows–Wheeler transform variant of Bauer et al. (a.k.a., BCR BWT) in linear expected time. Our method uses compression techniques to reduce computational costs when the input is massive and repetitive. Concretely, we build on induced suffix sorting (ISS) and resort to run-length and grammar compression to maintain our intermediate results in compact form. Our compression format not only saves space but also speeds up the required computations. Our experiments show important space and computation time savings when the text is repetitive. In moderate-size collections of real human genome assemblies (14.2 GB - 75.05 GB), our memory peak is, on average, 1.7x smaller than the peak of the state-of-the-art BCR BWT construction algorithm (ropebwt2), while running 5x faster. Our current implementation was also able to compute the BCR BWT of 400 real human genome assemblies (1.2 TB) in 41.21 hours using 118.83 GB of working memory (around 10% of the input size). Interestingly, the results we report in the 1.2 TB file are dominated by the difficulties of scanning huge files under memory constraints (specifically, I/O operations). This fact indicates we can perform much better with a more careful implementation of our method, thus scaling to even bigger sizes efficiently.

Benchmarking multi-platform sequencing technologies for human genome assembly.

Genome assembly is a computational technique that involves piecing together deoxyribonucleic acid (DNA) fragments generated by sequencing technologies to create a comprehensive and precise representation of the entire genome. Generating a high-quality human reference genome is a crucial prerequisite for comprehending human biology, and it is also vital for downstream genomic variation analysis. Many efforts have been made over the past few decades to create a complete and gapless reference genome for humans by using a diverse range of advanced sequencing technologies. Several available tools are aimed at enhancing the quality of haploid and diploid human genome assemblies, which include contig assembly, polishing of contig errors, scaffolding and variant phasing. Selecting the appropriate tools and technologies remains a daunting task despite several studies have investigated the pros and cons of different assembly strategies. The goal of this paper was to benchmark various strategies for human genome assembly by combining sequencing technologies and tools on two publicly available samples (NA12878 and NA24385) from Genome in a Bottle. We then compared their performances in terms of continuity, accuracy, completeness, variant calling and phasing. We observed that PacBio HiFi long-reads are the optimal choice for generating an assembly with low base errors. On the other hand, we were able to produce the most continuous contigs with Oxford Nanopore long-reads, but they may require further polishing to improve on quality. We recommend using short-reads rather than long-reads themselves to improve the base accuracy of contigs from Oxford Nanopore long-reads. Hi-C is the best choice for chromosome-level scaffolding because it can capture the longest-range DNA connectedness compared to 10× linked-reads and Bionano optical maps. However, a combination of multiple technologies can be used to further improve the quality and completeness of genome assembly. For diploid assembly, hifiasm is the best tool for human diploid genome assembly using PacBio HiFi and Hi-C data. Looking to the future, we expect that further advancements in human diploid assemblers will leverage the power of PacBio HiFi reads and other technologies with long-range DNA connectedness to enable the generation of high-quality, chromosome-level and haplotype-resolved human genome assemblies.

Lost in translation: the pitfalls of Ensembl gene annotations between human genome assemblies and their impact on diagnostics

ABSTRACT Background Gene models based on GRCh37 human genome assembly are preferred by many international projects over other updated assemblies (GRCh38 and T2T). Discrepant genes (DGs), those recognized as protein coding in the new but not the old assembly, are ignored by several genomic resources and discarded by variant prioritization tools relying on information based on GRCh37. Methods We curated a set of Ensembl genes with discrepant annotations between GRCh37 and GRCh38, additionally matching their RefSeq transcripts. Furthermore, we examined their clinical and phenotypic relevance. Results A total of 337 genes were reclassified as ‘protein-coding’ in GRCh38 but not in GRCh37, with 194 having a discrepant HGNC gene symbol. Many remain missing from the current known RefSeq gene models (N = 73). We found many clinically relevant genes in this group of neglected genes, and we anticipate that many more will be found relevant in the future. Important additional annotations such as evolutionary constraint metrics are also not calculated for these genes, further relegating them into oblivion. Conclusion For discrepant genes, the inaccurate label of ‘non-protein-coding’ has relevant ramifications on clinical genetics. Accurate collation of these genes allows for manual curation in clinically relevant scenarios.