LRTK: a platform agnostic toolkit for linked-read analysis of both human genome and metagenome.

BACKGROUND: Linked-read sequencing technologies generate high-base quality short reads that contain extrapolative information on long-range DNA connectedness. These advantages of linked-read technologies are well known and have been demonstrated in many human genomic and metagenomic studies. However, existing linked-read analysis pipelines (e.g., Long Ranger) were primarily developed to process sequencing data from the human genome and are not suited for analyzing metagenomic sequencing data. Moreover, linked-read analysis pipelines are typically limited to 1 specific sequencing platform. FINDINGS: To address these limitations, we present the Linked-Read ToolKit (LRTK), a unified and versatile toolkit for platform agnostic processing of linked-read sequencing data from both human genome and metagenome. LRTK provides functions to perform linked-read simulation, barcode sequencing error correction, barcode-aware read alignment and metagenome assembly, reconstruction of long DNA fragments, taxonomic classification and quantification, and barcode-assisted genomic variant calling and phasing. LRTK has the ability to process multiple samples automatically and provides users with the option to generate reproducible reports during processing of raw sequencing data and at multiple checkpoints throughout downstream analysis. We applied LRTK on linked reads from simulation, mock community, and real datasets for both human genome and metagenome. We showcased LRTK's ability to generate comparative performance results from preceding benchmark studies and to report these results in publication-ready HTML document plots. CONCLUSIONS: LRTK provides comprehensive and flexible modules along with an easy-to-use Python-based workflow for processing linked-read sequencing datasets, thereby filling the current gap in the field caused by platform-centric genome-specific linked-read data analysis tools.

Range-limited Heaps' law for functional DNA words in the human genome.

Heaps' or Herdan-Heaps' law is a linguistic law describing the relationship between the vocabulary/dictionary size (type) and word counts (token) to be a power-law function. Its existence in genomes with certain definition of DNA words is unclear partly because the dictionary size in genome could be much smaller than that in a human language. We define a DNA word as a coding region in a genome that codes for a protein domain. Using human chromosomes and chromosome arms as individual samples, we establish the existence of Heaps' law in the human genome within limited range. Our definition of words in a genomic or proteomic context is different from other definitions such as over-represented k-mers which are much shorter in length. Although an approximate power-law distribution of protein domain sizes due to gene duplication and the related Zipf's law is well known, their translation to the Heaps' law in DNA words is not automatic. Several other animal genomes are shown herein also to exhibit range-limited Heaps' law with our definition of DNA words, though with various exponents. When tokens were randomly sampled and sample sizes reach to the maximum level, a deviation from the Heaps' law was observed, but a quadratic regression in log-log type-token plot fits the data perfectly. Investigation of type-token plot and its regression coefficients could provide an alternative narrative of reusage and redundancy of protein domains as well as creation of new protein domains from a linguistic perspective.

Analysis of CENP-B Boxes as Anchor of Kinetochores in Centromeres of Human Chromosomes.

The kinetochore is a multiprotein structure that attaches at one end to DNA in the centromere and at the other end to microtubules in the mitotic spindle. By connecting centromere and spindle, the kinetochore controls the migration of chromosomes during cell division. The exact position where the kinetochore assembles on each centromere was uncertain because large sections of centromeric DNA had not been sequenced due to highly repetitive alpha-satellite arrays. Embedded in the arrays is a 17 bp consensus sequence, the so-called CENP-B box, which binds the CENP-B protein, the only protein that binds directly to centromeric DNA. Recently, the Telomere-to-Telomere Consortium published the complete centromeric DNA sequences of all chromosomes including their epigenetic modifications in the T2T-CHM13 map. I used data from the T2T-CHM13 map to locate the CENP-B boxes in the centromeres as anchor of kinetochores. Most of the CENP-B boxes in centromeric DNA are methylated with the exception of the so-called centromere dip region (CDR), where CENP-B protein dimers bind to adjacent unmethylated CENP-B boxes and interact with CENP-A and CENP-C proteins to assemble the kinetochore. The centromeres of all chromosomes combined have a size of 407 Mb of which the kinetochores account for 5.0 Mb or 1.2%. There is no correlation between centromere and kinetochore size (P = .77). While the number of CENP-B boxes varies 4-fold between chromosomes, their density (number/Kb) varies less than 2-fold with a mean of 2.61 ± 0.33. The narrow range ensures a uniform pull of the spindle on the centromeres. I illustrate the findings in a model of the human kinetochore anchored at unmethylated CENP-B boxes in the CDR and present circos plots of chromosomes to show the location of kinetochores in their respective centromeres.

Convergent Mutations and Single Nucleotide Variants in Mitochondrial Genomes of Modern Humans and Neanderthals.

The genetic contributions of Neanderthals to the modern human genome have been evidenced by the comparison of present-day human genomes with paleogenomes. Neanderthal signatures in extant human genomes are attributed to intercrosses between Neanderthals and archaic anatomically modern humans (AMHs). Although Neanderthal signatures are well documented in the nuclear genome, it has been proposed that there is no contribution of Neanderthal mitochondrial DNA to contemporary human genomes. Here we show that modern human mitochondrial genomes contain 66 potential Neanderthal signatures, or Neanderthal single nucleotide variants (N-SNVs), of which 36 lie in coding regions and 7 result in nonsynonymous changes. Seven N-SNVs are associated with traits such as cycling vomiting syndrome, Alzheimer's disease and Parkinson's disease, and two N-SNVs are associated with intelligence quotient. Based on recombination tests, principal component analysis (PCA) and the complete absence of these N-SNVs in 41 archaic AMH mitogenomes, we conclude that convergent evolution, and not recombination, explains the presence of N-SNVs in present-day human mitogenomes.

Advancing human genotyping: The Infinium HTS iSelect Custom microarray panel (Rita) development study.

Single Nucleotide Polymorphisms (SNPs), as the most prevalent type of variation in the human genome, play a pivotal role in influencing human traits. They are extensively utilized in diverse fields such as population genetics, forensic science, and genetic medicine. This study focuses on the 'Rita' BeadChip, a custom SNP microarray panel developed using Illumina Infinium HTS technology. Designed for high-throughput genotyping, the panel facilitates the analysis of over 4000 markers efficiently and cost-effectively. After careful clustering performed on a set of 1000 samples, an evaluation of the Rita panel was undertaken, assessing its sensitivity, repeatability, reproducibility, precision, accuracy, and resistance to contamination. The panel's performance was evaluated in various scenarios, including sex estimation and parental relationship assessment, using GenomeStudio data analysis software. Findings show that over 95 % of the custom BeadChip assay markers were successful, with better performance of transitions over other mutations, and a considerably lower success rate for Y chromosome loci. An exceptional call rate exceeding 99 % was demonstrated for control samples, even with DNA input as low as 0.781 ng. Call rates above 80 % were still obtained with DNA quantities under 0.1 ng, indicating high sensitivity and suitability for forensic applications where DNA quantity is often limited. Repeatability, reproducibility, and precision studies revealed consistency of the panel's performance across different batches and operators, with no significant deviations in call rates or genotyping results. Accuracy assessments, involving comparison with multiple available genetic databases, including the 1000 Genome Project and HapMap, denoted over 99 % concordance, establishing the Rita panel's reliability in genotyping. The contamination study revealed insights into background noise and allowed the definition of thresholds for sample quality evaluation. Multiple metrics for differentiating between negative controls and true samples were highlighted, increasing the reliability of the obtained results. The sex estimation tool in GenomeStudio proved highly effective, correctly assigning sex in all samples with autosomal loci call rates above 97 %. The parental relationship assessment of family trios highlighted the utility of GenomeStudio in identifying genotyping errors or potential Mendelian inconsistencies, promoting the application of arrays such as Rita in kinship testing. Overall, this evaluation confirms the Rita microarray as a robust, high-throughput genotyping tool, underscoring its potential in genetic research and forensic applications. With its custom content and adaptable design, it not only meets current genotyping demands but also opens avenues for further research and application expansion in the field of genetic analysis.

Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

Benchmarking long-read aligners and SV callers for structural variation detection in Oxford nanopore sequencing data.

Structural variants (SVs) are one of the significant types of DNA mutations and are typically defined as larger-than-50-bp genomic alterations that include insertions, deletions, duplications, inversions, and translocations. These modifications can profoundly impact the phenotypic characteristics and contribute to disorders like cancer, response to treatment, and infections. Four long-read aligners and five SV callers have been evaluated using three Oxford Nanopore NGS human genome datasets in terms of precision, recall, and F1-score statistical metrics, depth of coverage, and speed of analysis. The best SV caller regarding recall, precision, and F1-score when matched with different aligners at different coverage levels tend to vary depending on the dataset and the specific SV types being analyzed. However, based on our findings, Sniffles and CuteSV tend to perform well across different aligners and coverage levels, followed by SVIM, PBSV, and SVDSS in the last place. The CuteSV caller has the highest average F1-score (82.51%) and recall (78.50%), and Sniffles has the highest average precision value (94.33%). Minimap2 as an aligner and Sniffles as an SV caller act as a strong base for the pipeline of SV calling because of their high speed and reasonable accomplishment. PBSV has a lower average F1-score, precision, and recall and may generate more false positives and overlook some actual SVs. Our results are valuable in the comprehensive evaluation of popular SV callers and aligners as they provide insight into the performance of several long-read aligners and SV callers and serve as a reference for researchers in selecting the most suitable tools for SV detection.

Heritable human genome editing: correction, selection and treatment.

Heritable human genome editing (HHGE) to correct a nuclear gene sequence that would result in a serious genetic condition in a future child is presented as 'treatment' in various ethics and policy materials, and as morally preferable to the 'selection' practice of preimplantation genetic testing (PGT), which is subject to the disability critique. However, whether HHGE is 'treatment' for a future child, or another form of 'selection', or whether HHGE instead 'treats' prospective parents, are now central questions in the debate regarding its possible legalisation. This article argues that the idea of 'treatment' for a future child is largely a proxy for 'seriousness of purpose', intended to distinguish HHGE to avoid serious genetic conditions from less obviously justifiable uses; that HHGE is best understood, and morally justified, as a form of 'treatment' for prospective parents who strongly desire an unaffected genetically related child and who have no, or poor, options to achieve this; that HHGE would be morally permissible if consistent with that child's welfare; that legalisation is supportable with reference to the right to respect for private and family life under Article 8 of the European Convention on Human Rights; and that HHGE is morally distinguishable from PGT.

Public participation in human genome editing research governance: what do scientists think?

Within the numerous policy and governance recommendations for human genome editing research, anticipatory public engagement seems universally agreed upon as a vital endeavor. Yet it is unclear whether and how scientists whose research involves genome editing see value in engaging the public in discussions of genome editing research governance. To address this question, we interviewed 81 international scientists who use genome editing in their research. The views of our scientist interviewees about public engagement occupied a broad spectrum from enthusiastic support to strong skepticism. But most scientists' views landed somewhere in the middle, seeing public engagement as merely informing the public about the science of genome editing. We argue that such a stance reflects the traditional "knowledge-deficit model." Beyond addressing the operational difficulties of public engagement, many scientists' adherence to the deficit model is a deeper barrier that needs to be addressed if public engagement is to occur and be successful.

Are non-protein coding RNAs junk or treasure?: An attempt to explain and reconcile opposing viewpoints of whether the human genome is mostly transcribed into non-functional or functional RNAs.

The human genome project's lasting legacies are the emerging insights into human physiology and disease, and the ascendance of biology as the dominant science of the 21st century. Sequencing revealed that >90% of the human genome is not coding for proteins, as originally thought, but rather is overwhelmingly transcribed into non-protein coding, or non-coding, RNAs (ncRNAs). This discovery initially led to the hypothesis that most genomic DNA is "junk", a term still championed by some geneticists and evolutionary biologists. In contrast, molecular biologists and biochemists studying the vast number of transcripts produced from most of this genome "junk" often surmise that these ncRNAs have biological significance. What gives? This essay contrasts the two opposing, extant viewpoints, aiming to explain their bases, which arise from distinct reference frames of the underlying scientific disciplines. Finally, it aims to reconcile these divergent mindsets in hopes of stimulating synergy between scientific fields.

The human PTGR1 gene expression is controlled by TE-derived Z-DNA forming sequence cooperating with miR-6867-5p.

Z-DNA, a well-known non-canonical form of DNA involved in gene regulation, is often found in gene promoters. Transposable elements (TEs), which make up 45% of the human genome, can move from one location to another within the genome. TEs play various biological roles in host organisms, and like Z-DNA, can influence transcriptional regulation near promoter regions. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a critical role in the regulation of gene expression. Although TEs can generate Z-DNA and miRNAs can bind to Z-DNA, how these factors affect gene transcription has yet to be elucidated. Here, we identified potential Z-DNA forming sequence (ZFS), including TE-derived ZFS, in the promoter of prostaglandin reductase 1 (PTGR1) by data analysis. The transcriptional activity of these ZFS in PTGR1 was confirmed using dual-luciferase reporter assays. In addition, we discovered a novel ZFS-binding miRNA (miR-6867-5p) that suppressed PTGR1 expression by targeting to ZFS. In conclusion, these findings suggest that ZFS, including TE-derived ZFS, can regulate PTGR1 gene expression and that miR-6867-5p can suppress PTGR1 by interacting with ZFS.

Survey of attitude to human genome modification in Nigeria.

Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30 years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.

Regulatory and governance gaps for human genome editing in Mexico.

Mexico has the in-house technical and regulatory capacity to undertake human genome editing (HGE) governance. However, its regulatory framework must be reformed to be more targeted and govern the application of any emerging HGE technologies, leaving no room for unethical or unsafe practices for reproductive purposes.

Expanding horizons of tandem repeats in biology and medicine: Why 'genomic dark matter' matters.

Approximately half of the human genome includes repetitive sequences, and these DNA sequences (as well as their transcribed repetitive RNA and translated amino-acid repeat sequences) are known as the repeatome. Within this repeatome there are a couple of million tandem repeats, dispersed throughout the genome. These tandem repeats have been estimated to constitute ∼8% of the entire human genome. These tandem repeats can be located throughout exons, introns and intergenic regions, thus potentially affecting the structure and function of tandemly repetitive DNA, RNA and protein sequences. Over more than three decades, more than 60 monogenic human disorders have been found to be caused by tandem-repeat mutations. These monogenic tandem-repeat disorders include Huntington's disease, a variety of ataxias, amyotrophic lateral sclerosis and frontotemporal dementia, as well as many other neurodegenerative diseases. Furthermore, tandem-repeat disorders can include fragile X syndrome, related fragile X disorders, as well as other neurological and psychiatric disorders. However, these monogenic tandem-repeat disorders, which were discovered via their dominant or recessive modes of inheritance, may represent the 'tip of the iceberg' with respect to tandem-repeat contributions to human disorders. A previous proposal that tandem repeats may contribute to the 'missing heritability' of various common polygenic human disorders has recently been supported by a variety of new evidence. This includes genome-wide studies that associate tandem-repeat mutations with autism, schizophrenia, Parkinson's disease and various types of cancers. In this article, I will discuss how tandem-repeat mutations and polymorphisms could contribute to a wide range of common disorders, along with some of the many major challenges of tandem-repeat biology and medicine. Finally, I will discuss the potential of tandem repeats to be therapeutically targeted, so as to prevent and treat an expanding range of human disorders.

The Human Genome Organisation (HUGO) and a vision for Ecogenomics: the Ecological Genome Project.

BACKGROUND: The following outlines ethical reasons for widening the Human Genome Organisation's (HUGO) mandate to include ecological genomics. MAIN: The environment influences an organism's genome through ambient factors in the biosphere (e.g. climate and UV radiation), as well as the agents it comes into contact with, i.e. the epigenetic and mutagenic effects of inanimate chemicals and pollution, and pathogenic organisms. Emerging scientific consensus is that social determinants of health, environmental conditions and genetic factors work together to influence the risk of many complex illnesses. That paradigm can also explain the environmental and ecological determinants of health as factors that underlie the (un)healthy ecosystems on which communities rely. We suggest that The Ecological Genome Project is an aspirational opportunity to explore connections between the human genome and nature. We propose consolidating a view of Ecogenomics to provide a blueprint to respond to the environmental challenges that societies face. This can only be achieved by interdisciplinary engagement between genomics and the broad field of ecology and related practice of conservation. In this respect, the One Health approach is a model for environmental orientated work. The idea of Ecogenomics-a term that has been used to relate to a scientific field of ecological genomics-becomes the conceptual study of genomes within the social and natural environment. CONCLUSION: The HUGO Committee on Ethics, Law and Society (CELS) recommends that an interdisciplinary One Health approach should be adopted in genomic sciences to promote ethical environmentalism. This perspective has been reviewed and endorsed by the HUGO CELS and the HUGO Executive Board.

The human genome as the common heritage of humanity.

While debate on the international regulation of human genomic research remains unsettled, the Universal Declaration on the Human Genome and Human Rights, 1997 qualifies the human genome as "heritage of humankind" in a symbolic sense. Using document analysis this article assesses whether, how and to what extent the common heritage framework is relevant in regulation of human genomic research. The article traces the history of the Human Genome Project to reveal the international community's race against privatization of the human genome and its resulting qualification as the common heritage of humanity. Further, it reviews the archival records of UNESCO's International Bioethics Committee to discover the rationale for qualifying the human genome as common heritage of humankind. The article finds that the common heritage of mankind framework remains relevant to the application of the human genome at the collective level. However, the framework is at odds with the individual dimension of the human genome based on individual personality rights. The article thus argues that the right to benefit from scientific progress and its applications offers an alternative international regulatory framework for human genomic research.

Analysis of gut microbiome, host genetics, and plasma metabolites reveals gut microbiome-host interactions in the Japanese population.

Interaction between the gut microbiome and host plays a key role in human health. Here, we perform a metagenome shotgun-sequencing-based analysis of Japanese participants to reveal associations between the gut microbiome, host genetics, and plasma metabolome. A genome-wide association study (GWAS) for microbial species (n = 524) identifies associations between the PDE1C gene locus and Bacteroides intestinalis and between TGIF2 and TGIF2-RAB5IF gene loci and Bacteroides acidifiaciens. In a microbial gene ortholog GWAS, agaE and agaS, which are related to the metabolism of carbohydrates forming the blood group A antigen, are associated with blood group A in a manner depending on the secretor status determined by the East Asian-specific FUT2 variant. A microbiome-metabolome association analysis (n = 261) identifies associations between bile acids and microbial features such as bile acid metabolism gene orthologs including bai and 7ß-hydroxysteroid dehydrogenase. Our publicly available data will be a useful resource for understanding gut microbiome-host interactions in an underrepresented population.

Ethical challenges of conducting and reviewing human genomics research in Malaysia: An exploratory study.

Even though there is a significant amount of scholarly work examining the ethical issues surrounding human genomics research, little is known about its footing in Malaysia. This study aims to explore the experience of local researchers and research ethics committee (REC) members in developing it in Malaysia. In-depth interviews were conducted from April to May 2021, and the data were thematically analysed. In advancing this technology, both genomics researchers and REC members have concerns over how this research is being developed in the country especially the absence of a clear ethical and regulatory framework at the national level as a guidance. However, this study argues that it is not a salient issue as there are international guidelines in existence and both researchers and RECs will benefit from a training on the guidelines to ensure genomics research can be developed in an ethical manner.

Unveiling human origins of replication using deep learning: accurate prediction and comprehensive analysis.

Accurate identification of replication origins (ORIs) is crucial for a comprehensive investigation into the progression of human cell growth and cancer therapy. Here, we proposed a computational approach Ori-FinderH, which can efficiently and precisely predict the human ORIs of various lengths by combining the Z-curve method with deep learning approach. Compared with existing methods, Ori-FinderH exhibits superior performance, achieving an area under the receiver operating characteristic curve (AUC) of 0.9616 for K562 cell line in 10-fold cross-validation. In addition, we also established a cross-cell-line predictive model, which yielded a further improved AUC of 0.9706. The model was subsequently employed as a fitness function to support genetic algorithm for generating artificial ORIs. Sequence analysis through iORI-Euk revealed that a vast majority of the created sequences, specifically 98% or more, incorporate at least one ORI for three cell lines (Hela, MCF7 and K562). This innovative approach could provide more efficient, accurate and comprehensive information for experimental investigation, thereby further advancing the development of this field.

Transcriptome driven discovery of novel candidate genes for human neurological disorders in the telomer-to-telomer genome assembly era.

BACKGROUND: With the first complete draft of a human genome, the Telomere-to-Telomere Consortium unlocked previously concealed genomic regions for genetic analyses. These regions harbour nearly 2000 potential novel genes with unknown function. In order to uncover candidate genes associated with human neurological pathologies, a comparative transcriptome study using the T2T-CHM13 and the GRCh38 genome assemblies was conducted on previously published datasets for eight distinct human neurological disorders. RESULTS: The analysis of differential expression in RNA sequencing data led to the identification of 336 novel candidate genes linked to human neurological disorders. Additionally, it was revealed that, on average, 3.6% of the differentially expressed genes detected with the GRCh38 assembly may represent potential false positives. Among the noteworthy findings, two novel genes were discovered, one encoding a pore-structured protein and the other a highly ordered ß-strand-rich protein. These genes exhibited upregulation in multiple epilepsy datasets and hold promise as candidate genes potentially modulating the progression of the disease. Furthermore, an analysis of RNA derived from white matter lesions in multiple sclerosis patients indicated significant upregulation of 26 rRNA encoding genes. Additionally, putative pathology related genes were identified for Alzheimer's disease, amyotrophic lateral sclerosis, glioblastoma, glioma, and conditions resulting from the m.3242 A > G mtDNA mutation. CONCLUSION: The results presented here underline the potential of the T2T-CHM13 assembly in facilitating the discovery of candidate genes from transcriptome data in the context of human disorders. Moreover, the results demonstrate the value of remapping sequencing data to a superior genome assembly. Numerous potential pathology related genes, either as causative factors or related elements, have been unveiled, warranting further experimental validation.