Interspersed Repeats Research Articles

Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes.

Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus. Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased, with the available genomes of varying accuracy and completeness. We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species; Citrus australis (round lime) using highly accurate PacBio HiFi long reads, complemented with Hi-C scaffolding. Hifiasm with Hi-C integrated assembly resulted in a 331Mb genome of C. australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3Mb and 98.8% genome assembly completeness (BUSCO). Repeat analysis showed that more than 50% of the genome contained interspersed repeats. Among them, LTR elements were the predominant type (21.0%), of which LTR Gypsy (9.8%) and LTR copia (7.7%) elements were the most abundant repeats. A total of 29 464 genes and 32 009 transcripts were identified in the genome. Of these, 28 222 CDS (25 753 genes) had BLAST hits and 21 401 CDS (75.8%) were annotated with at least one GO term. Citrus specific genes for antimicrobial peptides, defense, volatile compounds and acidity regulation were identified. The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2, 4, 7 and 8. This chromosome scale, and haplotype resolved C. australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.

Genetic Diversity and Population Structure of Mycobacterium bovis at the Human-Animal-Ecosystem Interface in France: "A One Health Approach".

Mycobacterium bovis infects cattle and wildlife, and also causes a small proportion of tuberculosis cases in humans. In most European countries, M. bovis infections in cattle have been drastically reduced, but not eradicated. Here, to determine the M. bovis circulation within and between the human, cattle, and wildlife compartments, we characterized by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing the genetic diversity of M. bovis isolates collected from humans, cattle, and wildlife in France from 2000 to 2010. We also assessed their genetic structure within and among the different host groups, and across time and space. The M. bovis genetic structure and its spatiotemporal variations showed different dynamics in the human and animal compartments. Most genotypes detected in human isolates were absent in cattle and wildlife isolates, possibly because in patients, M. bovis infection was contracted abroad or was the reactivation of an old lesion. Therefore, they did not match the genetic pool present in France during the study period. However, some human-cattle exchanges occurred because some genotypes were common to both compartments. This study provides new elements for understanding M. bovis epidemiology in France, and calls for increased efforts to control this pathogen worldwide.

Phylogenetic relationships of Mycobacterium tuberculosis isolates in Poland: The emergence of Beijing genotype among multidrug-resistant cases.

The epidemiological situation of tuberculosis (TB) in Poland urges for its continuous and scrupulous monitoring. The objective of this study was to explore the genetic diversity of multidrug-resistant (MDR) and drug-susceptible (DS) Mycobacterium tuberculosis isolates from Poland with a combination of spoligotyping and high-resolution mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis. The results were placed in the Northern and Eastern Europe context. The study included 89 (39 MDR and 50 DS) M. tuberculosis isolates collected from as many patients between 2018 and 2021 in Poland. The analysis was done using spoligotyping, and MIRU-VNTR typing at 24 standard loci. The data were compared to those available on Poland and neighbors and global M. tuberculosis datasets. The main identified families were Beijing (28.1%) and Haarlem (16.8%) while 34.8% of isolates were in the heterogeneous L4-unclassified group. Although the Beijing family was the most prevalent (61.5%) among MDR-TB cases, it accounted for only 2% of DS isolates. Among foreign-born patients, a higher ratio of MDR isolates were observed when compared with those who Poland-born (64.3% vs. 40%). Furthermore, all patients from the Former Soviet Union (FSU) countries were infected with MDR-TB. Whereas DS M. tuberculosis population in Poland is dominated by L4 isolates, MDR isolates are mostly of the Beijing genotype. The rise in the prevalence of the Beijing isolates in Poland, coupled with high proportion of the Beijing genotype among foreign-born TB patients may reflect an ongoing transmission of this family, imported to Poland mainly from FSU countries.

Complete genome sequence analysis of Edwardsiella tarda SC002 from hatchlings of Siamese crocodile.

Edwardsiella tarda is a Gram-negative, facultative anaerobic rod-shaped bacterium and the causative agent of the systemic disease "Edwardsiellosis". It is commonly prevalent in aquatic organisms with subsequent economic loss and hence has attracted increasing attention from researchers. In this study, we investigated the complete genome sequence of a highly virulent isolate Edwardsiella tarda SC002 isolated from hatchlings of the Siamese crocodile. The genome of SC002 consisted of one circular chromosome of length 3,662,469 bp with a 57.29% G+C content and four novel plasmids. A total of 3,734 protein-coding genes, 12 genomic islands (GIs), 7 prophages, 48 interspersed repeat sequences, 248 tandem repeat sequences, a CRISPR component with a total length of 175 bp, and 171 ncRNAs (tRNA = 106, sRNA = 37, and rRNA = 28) were predicted. In addition, the coding genes of assembled genome were successfully annotated against eight general databases (NR = 3,618/3,734, COG = 2,947/3,734, KEGG = 3,485/3,734, SWISS-PROT = 2,787/3,734, GO = 2,648/3,734, Pfam = 2,648/3,734, CAZy = 130/3,734, and TCDB = 637/3,734) and four pathogenicity-related databases (ARDB = 11/3,734, CARD = 142/3,734, PHI = 538/3,734, and VFDB = 315/3,734). Pan-genome and comparative genome analyses of the complete sequenced genomes confirmed their evolutionary relationships. The present study confirmed that E. tarda SC002 is a potential pathogen bearing a bulk amount of antibiotic resistance, virulence, and pathogenic genes and its open pan-genome may enhance its host range in the future.

Transcriptome analysis of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) and characterization of genes associated to immunity, growth, and reproduction

BackgroundDung beetles recycle organic matter through the decomposition of feces and support ecological balance. However, these insects are threatened by the indiscriminate use of agrochemicals and habitat destruction. Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae), a dung beetle, is listed as a class-II Korean endangered species. Although the genetic diversity of C. tripartitus populations has been investigated through analysis of mitochondrial genes, genomic resources for this species remain limited. In this study, we analyzed the transcriptome of C. tripartitus to elucidate functions related to growth, immunity and reproduction for the purpose of informed conservation planning.ResultsThe transcriptome of C. tripartitus was generated using next-generation Illumina sequencing and assembled de novo using a Trinity-based platform. In total, 98.59% of the raw sequence reads were processed as clean reads. These reads were assembled into 151,177 contigs, 101,352 transcripts, and 25,106 unigenes. A total of 23,450 unigenes (93.40%) were annotated to at least one database. The largest proportion of unigenes (92.76%) were annotated to the locally curated PANM-DB. A maximum of 5,512 unigenes had homologous sequences in Tribolium castaneum. Gene Ontology (GO) analysis revealed a maximum of 5,174 unigenes in the Molecular function category. Further, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, a total of 462 enzymes were associated with established biological pathways. Based on sequence homology to known proteins in PANM-DB, representative immunity, growth, and reproduction-related genes were screened. Potential immunity-related genes were categorized into pattern recognition receptors (PRRs), the Toll-like receptor signaling pathway, the MyD88- dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related transcripts. Among PRRs, we conducted detailed in silico characterization of TLR-2, CTL, and PGRP_SC2-like. Repetitive elements such as long terminal repeats, short interspersed nuclear elements, long interspersed nuclear elements and DNA elements were enriched in the unigene sequences. A total of 1,493 SSRs were identified among all unigenes of C. tripartitus.ConclusionsThis study provides a comprehensive resource for analysis of the genomic topography of the beetle C. tripartitus. The data presented here clarify the fitness phenotypes of this species in the wild and provide insight to support informed conservation planning.

Dual-Responsive Core-Shell Tecto Dendrimers Enable Efficient Gene Editing of Cancer Cells to Boost Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy has become a promising strategy in treating multiple tumor types, but the therapeutic efficacy is still unsatisfactory due to the temporary and inefficient blocking and the poor immune responsiveness. Herein, we report the development of dual reactive oxygen species (ROS)- and pH-responsive core-shell tecto dendrimers loaded with gold nanoparticles (for short, Au CSTDs) to deliver a plasmid-clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system for the permanent disruption of the programmed death ligand 1 (PD-L1) gene in cancer cells to boost cancer immunotherapy. In our work, Au CSTDs were constructed using lactobionic acid (LA)-modified generation 5 poly(amidoamine) dendrimers entrapped with gold nanoparticles as cores and phenylboronic acid (PBA)-conjugated generation 3 dendrimers as shells via the formation of responsive phenylborate ester bonds between PBA and LA. The plasmid-CRISPR/Cas9 system can be efficiently compacted and specifically taken up by cancer cells overexpressing sialic acids due to the PBA-mediated targeting and be responsively released in cancer cells by the responsive dissociation of the Au CSTDs, leading to the successful endosomal escape and the efficient knockout of the PD-L1 gene. Further in vivo delivery in a mouse melanoma model reveals that the developed Au CSTDs/plasmid-CRISPR/Cas9 complexes can be specifically accumulated at the tumor site for enhanced computed tomography (CT) imaging of tumors, owing to the X-ray attenuation effect of Au, and disrupt the PD-L1 expression in tumor cells, thus promoting the ICB-based antitumor immunity. The designed dual-responsive Au CSTDs may be developed as a versatile tool for genetic engineering of other cell types to achieve different therapeutic effects for expanded space of biomedical applications.

A Review of CRISPR Cas9 for SCA: Treatment Strategies and Could Target β-globin Gene and BCL11A Gene using CRISPR Cas9 Prevent the Patient from Sickle Cell Anemia?

BACKGROUND: Sickle cell anemia is a hereditary globin chain condition that leads to hemolysis and persistent organ damage. Chronic hemolytic anemia, severe acute and chronic pain, and end-organ destruction occur throughout the lifespan of sickle cell anemia. SCD is associated with a higher risk of mortality. Genome editing with CRISPR-associated regularly interspersed short palindromic repeats (CRISPR/Cas9) have therapeutic potential for sickle cell anemia thala. AIM: This research aimed to see if using CRISPR/Cas9 to target β-globin gene is an effective therapeutic and if it has a long-term effect on Sickle Cell Anemia. METHODS: The method used in this study summarizes the article by looking for keywords that have been determined in the title and abstract. The authors used official guidelines from Science Direct, PubMed, Google Scholar, and Journal Molecular Biology to select full-text articles published within the last decade, prioritizing searches within the past 10 years. RESULTS: CRISPR/Cas9-mediated genome editing in clinical trials contributes to α-globin gene deletion correcting β-thalassemia through balanced α- and β-globin ratios and inhibiting disease progression. CONCLUSION: HBB and BCL11A targeting by CRISPR/Cas9 deletion effectively inactivate BCL11A, a repressor of fetal hemoglobin production. However, further research is needed to determine its side effects and safety.

Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization.

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

DNA methylation status of interspersed repetitive sequences in patients with migraine.

To analyse the methylation status of the Long Interspersed Nuclear Element-1 (LINE-1) and Short Interspersed Nuclear Element Alu (Alu) of peripheral blood mononuclear cells (PBMCs) from patients with migraine compared with healthy control subjects. This case-control study recruited patients with migraine without aura and age-matched healthy control subjects. PBMCs were purified from peripheral blood samples. Methylation levels and patterns of LINE-1 and Alu sequences were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences polymerase chain reaction. A total of 84 patients with migraine and 82 age-matched healthy controls were enrolled in the study. High levels of unmethylated cytosines in both the LINE-1 and Alu repetitive elements were observed in the migraine group compared with the control subjects. In addition, a significant difference was detected in the methylation level of LINE-1 between TT and CC genotype groups of the methylenetetrahydrofolate reductase gene. These results suggest that analysis of epigenetic biomarkers in PBMCs may help to identify patients at a higher risk of migraine development.

MITE infestation accommodated by genome editing in the germline genome of the ciliate Blepharisma

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

The genome of a Far Eastern isolate of Diaporthe caulivora, a soybean fungal pathogen.

Diaporthe caulivora is an economically important fungal pathogen and a causal agent of soybean stem canker and seed decay. Here, the genome of a Russian Far Eastern isolate of D. caulivora was sequenced, assembled, and announced. Assembly quality was enough for advanced annotation, including prediction of potential disease-related genes encoding virulence factors and molecular determinants contributing to pathogen-host selection, interactions, and adaptation. Comparative analysis of 15 Diaporthe species was conducted regarding general genome properties, collinearity, and proteomes, and included detailed investigation of interspersed repeats. A notable feature of this analysis is a high recombinant variability of Diaporthe genomes, determined by the number and distribution of interspersed repeats, which also proved to be responsible for the diversity of GC content and genome size. This variability is assumed the main determinant of the divergence of Diaporthe genomes. A Bayesian multi-gene phylogeny was inferred for the 15 Diaporthe species on the basis of twenty thousand polymorphic sites of > 100 orthologous genes using independently adjusted evolutionary models. This allowed for the most accurate determination of evolutionary relationships and species boundaries for effective reporting about these plant pathogens. The evidence, obtained by different genome analysis techniques, implies the host-independent evolution of Diaporthe species. KEY POINTS: • The genome of a Far Eastern isolate of D. caulivora was announced. • A high degree of recombinant variability determines genomic divergence in Diaporthe genus. • The multi-gene phylogeny implies host-independent evolution of Diaporthe species.

The mutation rate of rpoB gene showed an upward trend with the increase of MIRU10, MIRU39 and QUB4156 repetitive number

BackgroundMycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) is a frequently used typing method for identifying the Beijing genotype of Mycobacterium tuberculosis (Mtb), which is easily transformed into rifampicin (RIF) resistance. The RIF resistance of Mtb is considered to be highly related with the mutation of rpoB gene. Therefore, this study aimed to analyze the relationship between the repetitive number of MIRU loci and the mutation of rpoB gene.MethodsAn open-source whole-genome sequencing data of Mtb was used to detect the mutation of rpoB gene and the repetitive number of MIRU loci by bioinformatics methods. Cochran-Armitage analysis was performed to analyze the trend of the rpoB gene mutation rate and the repetitive number of MIRU loci.ResultsAmong 357 rifampicin-resistant tuberculosis (RR-TB), 304 strains with mutated rpoB genes were detected, and 6 of 67 rifampicin susceptible strains were detected mutations. The rpoB gene mutational rate showed an upward trend with the increase of MIRU10, MIRU39, QUB4156 and MIRU16 repetitive number, but only the repetitive number of MIRU10, MRIU39 and QUB4156 were risk factors for rpoB gene mutation. The Hunter-Gaston discriminatory index (HGDI) of MIRU10 (0.65) and QUB4156 (0.62) was high in the overall sample, while MIRU39 (0.39) and MIRU16 (0.43) showed a moderate discriminatory Power.ConclusionThe mutation rate of rpoB gene increases with the addition of repetitive numbers of MIRU10, QUB4156 and MIRU39 loci.

RNA-targeting strategies as a platform for ocular gene therapy.

Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.

Recent progress in the genotyping of bovine tuberculosis and its rapid diagnosis via nanoparticle-based electrochemical biosensors.

Bovine tuberculosis (bTB) is considered a worldwide infectious zoonotic disease. Mycobacterium bovis causes bTB disease. It is one of the Mycobacterium tuberculosis complex (MTBC) members. MTBC is a clonal complex of close relatives with approximately 99.95% similarity. M. bovis is a spillover pathogen that can transmit from animals to humans and rarely from humans to animals with contact. Genotyping techniques are important to discriminate and differentiate between MTBC species. Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) are widely used but they have some limitations. As an alternative, whole genome sequencing approaches have been utilized due to their high-resolution power. They are employed in typing M. bovis and explain the evolutionary and phylogenetic relationships between isolates. The control of bTB disease has attracted a large amount of attention. Rapid and proper diagnosis is necessary for monitoring the disease as an initial step for its control and treatment. Nanotechnology has a potential impact on the rapid diagnosis and treatment of bTB through the use of nanocarrier and metal nanoparticles (NPs). Special attention has been paid to voltammetric and impedimetric electrochemical strategies as facile, sensitive, and selective methods for the efficient detection of tuberculosis. The efficacy of these sensors is enhanced in the presence of NPs, which act as recognition and/or redox probes. Gold, silver, copper, cobalt, graphene, and magnetic NPs, as well as polypyrrole nanowires and multiwalled carbon nanotubes have been employed for detecting tuberculosis. Overall, NP-based electrochemical sensors represent a promising tool for the diagnosis of bTB.

Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector.

The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4-5months.

Alteration of interspersed repetitive sequence methylation in T lymphocytes of people living with HIV: A preliminary study

Alteration of interspersed repetitive sequence methylation in T lymphocytes of people living with HIV: A preliminary study

RAfilter: an algorithm for detecting and filtering false-positive alignments in repetitive genomic regions.

Telomere to telomere (T2T) assembly relies on the correctness of sequence alignments. However, the existing aligners tend to generate a high proportion of false-positive alignments in repetitive genomic regions which impedes the generation of T2T-level reference genomes for more important species. In this paper, we present an automatic algorithm called RAfilter for removing the false-positives in the outputs of existing aligners. RAfilter takes advantage of rare k-mers representing the copy-specific features to differentiate false-positive alignments from the correct ones. Considering the huge numbers of rare k-mers in large eukaryotic genomes, a series of high-performance computing techniques such as multi-threading and bit operation are used to improve the time and space efficiencies. The experimental results on tandem repeats and interspersed repeats show that RAfilter was able to filter 60%-90% false-positive HiFi alignments with almost no correct ones removed, while the sensitivities and precisions on ONT datasets were about 80% and 50% respectively.

Complete chloroplast genomes of 11 Sabia samples: Genomic features, comparative analysis, and phylogenetic relationship.

The genus Sabia is a woody climber belonging to the family Sabiaceae, order Proteales. Several species of this genus have been utilized as medicines for treating diseases, such as rheumatic arthritis, traumatism, hepatitis, etc. However, the lack of molecular data has prevented the accurate identification and refinement of taxonomic relationships in this genus. In this study, chloroplast genomes of 11 samples of the genus Sabia were assembled and analyzed. These chloroplast genomes showed a typical quadripartite structure and ranged in length from 160,956 to 162,209 bp. The structure of the genomes was found to be relatively conserved, with 130 genes annotated, including 85 coding genes, 37 tRNA genes, and eight rRNA genes. A total of 78-98 simple sequence repeats and 52-61 interspersed repeats were detected. Sequence alignment revealed 11 highly variable loci in chloroplast genomes. Among these loci, ndhF-ndhD achieved a remarkably higher resolution than the other regions. In addition, phylogenetic analysis indicated that Sect. Pachydiscus and Sect. Sabia of Sabia did not form two separate monophyletic groups. The divergence time calculated based on the Reltime method indicated that the evolutionary branches of Sabia and Meliosma started to form approximately 85.95 million years ago (Mya), and the species within Sabia began to diverge approximately 7.65 Mya. In conclusion, our study provides a basis for comprehensively exploring the phylogenetic relationships of Sabia. It also provides a methodological basis and data support for establishing a standardized and scientific identification system for this genus.

Inferring Protein-DNA Binding Profiles at Interspersed Repeats Using HiChIP and PAtChER.

Alignment of short-read sequencing data to interspersed genomic repeats, such as transposable elements, can be problematic. This is especially true for evolutionarily young elements, which have not sufficiently diverged from each other to produce distinct and uniquely mappable reads. Mapping difficulties pose a challenge for studying the portfolio of epigenetic modifications and other chromatin regulators that bind to transposons and dictate their activity, which are typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Since ChIP-seq requires chromatin fragmentation to achieve appropriate resolution, longer reads do not appreciably improve mappability. Here, we present an experimental and computational protocol that couples ChIP-seq with 3D genome folding information to produce protein binding profiles with dramatically increased coverage at interspersed repeats.