The quality and detection limits of mitochondrial heteroplasmy by long read nanopore sequencing.

This study evaluates long-read and short-read sequencing for mitochondrial DNA (mtDNA) heteroplasmy detection. 592,315 bootstrapped datasets generated from two single-nucleotide mismatched ultra-deep sequenced mtDNA samples were used to assess basecalling error and accuracy, limit of heteroplasmy detection, and heteroplasmy detection across various coverage depths. Results showed high Phred scores of data with GC-rich sequence bias for long reads. Limit of detection of 12% heteroplasmy was identified, showing strong correlation (R2 ≥ 0.955) with expected heteroplasmy but underreporting tendency of high-level variants. Nanopore sequencing shows potential for direct applicability in mitochondrial diseases diagnostics, but stringent validation processes to ensure diagnostic result quality are required.

Delayed Diagnosis of Spinal Muscular Atrophy in Two Chinese Families due to Novel SMN1 Deletions.

Autosomal recessive spinal muscular atrophy (SMA) is a leading cause of infant and child mortality, with homozygous deletion in exon 7 of the SMN1 gene being a major genetic cause. However, routine genetic testing methods may overlook structural variants outside of exon 7, potentially leading to misdiagnosis of SMA patients. Here, we reported two Chinese SMA patients who primarily exhibited developmental delays. Physical examinations revealed markedly reduced muscle strength and tone in their extremities, and electromyography suggested extensive neurogenic damage in the anterior horn of the spinal cord. The MLPA results indicated a single copy number of SMN1 in both patients, which is inconsistent with the typical genetic pattern of SMA. Through RNA sequencing and ultra-long read sequencing, we ultimately identified a rare structural variant involving the deletion of exons 2a-5 in both unrelated patients. This confirmed the presence of compound heterozygous variants in the SMN1 gene as the actual genetic cause. To our knowledge, this is the first case where a combination of RNA sequencing and ultra-long read sequencing has been used to diagnose SMA. We demonstrated the significant value of RNA sequencing in cases where children are highly suspected of having SMA but present negative results in routine genetic testing. This underscores the crucial role of accurate genetic testing methods in achieving early diagnosis of SMA.

Assessing RNA integrity by digital RT-PCR: Influence of extraction, storage, and matrices.

The development of high-throughput sequencing has greatly improved our knowledge of microbial diversity in aquatic environments and its evolution in highly diverse ecosystems. Relevant microbial diversity description based on high-throughput sequencing relies on the good quality of the nucleic acid recovered. Indeed, long genetic fragments are more informative for identifying mutation combinations that characterize variants or species in complex samples. This study describes a new analytical method based on digital Polymerase Chain Reaction (PCR) partitioning technology for assessing the fragmentation of nucleic acid and more specifically viral RNA. This method allows us to overcome limits associated with hydrolysis probe-based assay by focusing on the distance between different amplicons, and not, as usual, on the size of amplicons. RNA integrity can thus be determined as a new fragmentation index, the so-called Fragment size 50. The application of this method has provided information on issues that are inherent in environmental analyses, such as the storage impact of raw samples or extracted RNA, extraction methods, and the nature of the sample on the integrity of viral RNA. Finally, the estimation of fragment size by digital PCR (dPCR) showed a very strong similarity with the fragment size sequenced using Oxford Nanopore Technology. In addition to enabling objective improvements in analytical methods, this approach could become a systematic quality control prior to any long-read sequencing, avoiding insufficiently productive sequencing runs or biases in the representativeness of sequenced fragments.

Chromosome-scale genome of the polyphagous pest Anastrepha ludens (Diptera: Tephritidae) provides insights on sex chromosome evolution in Anastrepha.

The Mexican fruit fly, Anastrepha ludens, is a polyphagous true fruit fly (Diptera: Tephritidae) considered one of the most serious insect pests in Central and North America to various economically relevant fruits. Despite its agricultural relevance, a high-quality genome assembly has not been reported. Here, we described the generation of a chromosome-level genome for the A. ludens using a combination of PacBio high fidelity long-reads and chromatin conformation capture sequencing data. The final assembly consisted of 140 scaffolds (821 Mb, N50 = 131 Mb), containing 99.27% complete conserved orthologs (BUSCO) for Diptera. We identified the sex chromosomes using three strategies: 1) visual inspection of Hi-C contact map and coverage analysis using the HiFi reads, 2) synteny with Drosophila melanogaster, and 3) the difference in the average read depth of autosomal versus sex chromosomal scaffolds. The X chromosome was found in one major scaffold (100 Mb) and eight smaller contigs (1.8 Mb), and the Y chromosome was recovered in one large scaffold (6.1 Mb) and 35 smaller contigs (4.3 Mb). Sex chromosomes and autosomes showed considerable differences of transposable elements and gene content. Moreover, evolutionary rates of orthologs of A. ludens and Anastrepha obliqua revealed a faster evolution of X-linked, compared to autosome-linked, genes, consistent with the faster-X effect, leading us to new insights on the evolution of sex chromosomes in this diverse group of flies. This genome assembly provides a valuable resource for future evolutionary, genetic, and genomic translational research supporting the management of this important agricultural pest.

Units containing telomeric repeats are prevalent in subtelomeric regions of a Mesorhabditis isolate collected from the Republic of Korea.

BACKGROUND: Mesorhabditis is known for its somatic genome being only a small portion of the germline genome due to programmed DNA elimination. This phenotype may be associated with the maintenance of telomeres at the ends of fragmented somatic chromosomes. OBJECTIVE: To comprehensively investigate the telomeric regions of Mesorhabditis nematodes at the sequence level, we endeavored to collect a Mesorhabditis nematode in the Republic of Korea and acquire its highly contiguous genome sequences. METHODS: We isolated a Mesorhabditis nematode and assembled its 108-Mb draft genome using both 6.3 Gb (53 ×) of short-read and 3.0 Gb (25 × , N50 = 5.7 kb) of nanopore-based long-read sequencing data. Our genome assembly exhibits comparable quality to the public genome of Mesorhabditis belari in terms of contiguity and evolutionary conserved genes. RESULTS: Unexpectedly, our Mesorhabditis genome has many more interstitial telomeric sequences (ITSs), specifically subtelomeric ones, compared to the genomes of Caenorhabditis elegans and M. belari. Moreover, several subtelomeric sequences containing ITSs had 4-26 homologous sequences, implying they are highly repetitive. Based on this highly repetitive nature, we hypothesize that subtelomeric ITSs might have accumulated through the action of transposable elements containing ITSs. CONCLUSIONS: It still remains elusive whether these ITS-containing units are associated with programmed DNA elimination, but they may facilitate new telomere formation after DNA elimination. Our genomic resources for Mesorhabditis can aid in understanding how its distinct phenotypes have evolved.

Complete genome sequences of five bacteria isolated from rice plants in a paddy field in Sekinchan, Selangor, Malaysia.

This study examines the genome sequences of five endophytic bacterial isolates from the Oryza sativa microbiome to assess their potential as plant bio-inoculants. The five complete bacterial genomes from the genera Pseudomonas, Burkholderia, Sphingobacterium, Stenotrophomonas, and Pantoea were sequenced using Nanopore long-read sequencing technology.

Promoter Deletion Leading to Allele Specific Expression in a Genetically Unsolved Case of Primary Ciliary Dyskinesia.

Variation in the non-coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non-coding genomic regions will be detrimental. Our approach using complementary RNA-seq and targeted long-read DNA sequencing can prioritize identification of non-coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non-coding variant on the second allele that impacts transcription. Targeted long-read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5' untranslated region of SPAG1, overlapping the promoter and first non-coding exon. This non-coding deletion was missed by whole exome sequencing and gene-specific deletion/duplication analysis, highlighting the importance of investigating the non-coding genome in patients with "missing" disease-causing variation. This paradigm demonstrates the utility of both RNA and long-read DNA sequencing in identifying pathogenic non-coding variants in patients with unexplained genetic disease.

3-hour genome sequencing and targeted analysis to rapidly assess genetic risk.

Purpose: Rapid genetic testing in the critical care setting may guide diagnostic evaluation, direct therapies, and help families and care providers make informed decisions about goals of care. We tested whether a simplified DNA extraction and library preparation process would enable us to perform ultra-rapid assessment of genetic risk for a Mendelian condition, based on information from an affected sibling, using long-read genome sequencing and targeted analysis. Methods: Following extraction of DNA from cord blood and rapid library preparation, genome sequencing was performed on an Oxford Nanopore PromethION. FASTQ files were generated from original sequencing data in near real-time and aligned to a reference genome. Variant calling and analysis were performed at timed intervals. Results: We optimized the DNA extraction and library preparation methods to create sufficient library for sequencing from 500 µL of blood. Real-time, targeted analysis was performed to determine that the newborn was neither affected nor a heterozygote for variants underlying a Mendelian condition. Phasing of the target region and prior knowledge of the affected haplotypes supported our interpretation despite a low level of coverage at 3 hours of life. Conclusion: This proof-of-concept experiment demonstrates how prior knowledge of haplotype structure or familial variants can be used to rapidly evaluate an individual at risk for a genetic disease. While ultra-rapid sequencing remains both complex and cost prohibitive, our method is more easily automated than prior approaches and uses smaller volumes of blood, thus may be more easily adopted for future studies of ultra-rapid genome sequencing in the clinical setting.

The complete genome sequence of five pre-2013 Escherichia coli sequence type (ST)1193 strains reveals insights into an emerging pathogen.

Fluoroquinolone-resistant Escherichia coli sequence type (ST)1193 is a profound, emerging lineage associated with systemic, urinary tract and neonatal infections. Humans, companion animals and the environment are reservoirs for ST1193, which has been disseminated globally. Following its detection in 2007, ST1193 has been identified repeatedly amongst fluoroquinolone-resistant clones in Australia. However, despite the growing importance of ST1193, only three complete genomes are published in the literature, none of which are from Australia. Here we expand on the available ST1193 resources with the complete genomes of five ST1193 strains sequenced using Oxford Nanopore Technologies and Illumina. Using in silico genotyping, we found that all strains were multi-drug resistant, including resistances to fluoroquinolones and cephalosporins. In vitro antibiotic susceptibility testing mostly correlated with individual genotypes. The exception was MS8320, which had additional in vitro resistance to piperacillin/tazobactam, ampicillin/sulbactam, cefazolin and doripenem (carbapenem). Further investigation identified seven additional copies of an IS26 transposable unit carrying a bla TEM-1B beta-lactamase gene, suggesting this tandem amplification is associated with extended resistance phenotypes. Uropathogenicity factors, including three separate siderophore-encoding loci, were conserved in chromosomal and plasmid regions. Using all complete genomes, we further elucidated the recombination events surrounding the previously described K5/K1 capsular locus switch. Phenotypic confirmation of differing capsules in Australian ST1193 strains, coupled with genetic analysis revealing insertions downstream of the capsular locus, underscored the genetic distinctions between K5 and K1 capsule encoding strains. This study provides five new reference ST1193 genomes from Australia. These include the earliest complete K5-capsule ST1193 genomes on record (collected 2007), alongside our reference genome (MS10858), a clinical isolate obtained early during the ST1193 expansion and representative of the predominant K1-associated clade. These findings lay the foundations for further genomic and molecular analyses that may help understand the underlying reasons for the rapid global expansion of ST1193.

Long-read sequencing and genome assembly of natural history collection samples and challenging specimens.

Museum collections harbor millions of samples, largely unutilized for long-read sequencing. Here, we use ethanol-preserved samples containing kilobase-sized DNA to show that amplification-free protocols can yield contiguous genome assemblies. Additionally, using a modified amplification-based protocol, employing an alternative polymerase to overcome PCR bias, we assembled the 3.1 Gb maned sloth genome, surpassing the previous 500 Mb protocol size limit. Our protocol also improves assemblies of other difficult-to-sequence molluscs and arthropods, including millimeter-sized organisms. By highlighting collections as valuable sample resources and facilitating genome assembly of tiny and challenging organisms, our study advances efforts to obtain reference genomes of all eukaryotes.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes.

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Characterising tandem repeat complexities across long-read sequencing platforms with TREAT and otter.

Tandem repeats (TR) play important roles in genomic variation and disease risk in humans. Long-read sequencing allows for the accurate characterization of TRs, however, the underlying bioinformatics perspectives remain challenging. We present otter and TREAT: otter is a fast targeted local assembler, cross-compatible across different sequencing platforms. It is integrated in TREAT, an end-to-end workflow for TR characterization, visualization and analysis across multiple genomes. In a comparison with existing tools based on long-read sequencing data from both Oxford Nanopore Technology (ONT, Simplex and Duplex) and PacBio (Sequel 2 and Revio), otter and TREAT achieved state-of-the-art genotyping and motif characterisation accuracy. Applied to clinically relevant TRs, TREAT/otter significantly identified individuals with pathogenic TR expansions. When applied to a case-control setting, we significantly replicated previously reported associations of TRs with Alzheimer's Disease, including those near or within APOC1 (p=2.63x10-9), SPI1 (p=6.5x10-3) and ABCA7 (p=0.04) genes. We used TREAT/otter to systematically evaluate potential biases when genotyping TRs using diverse ONT and PacBio long-read sequencing datasets. We showed that, in rare cases (0.06%), long-read sequencing suffers from coverage drops in TRs, including the disease-associated TRs in ABCA7 and RFC1 genes. Such coverage drops can lead to TR misgenotyping, hampering the accurate characterization of TR alleles. Taken together, our tools can accurately genotype TR across different sequencing technologies and with minimal requirements, allowing end-to-end analysis and comparisons of TR in human genomes, with broad applications in research and clinical fields.

Haplotypes analysis reveals the genetic basis of type I CD36 deficiency.

CD36, also known as glycoprotein IV, is classified into two distinct subgroups based on the presence or absence of its expression on monocytes. The CD36 gene spans approximately 50,000 base pairs. Historically, research has focused on identifying CD36 mutations through Sanger sequencing and next-generation sequencing (NGS), with limited exploration of haplotypes. In this study, we collected blood samples from donors with type I and type II CD36 deficiencies as well as from healthy controls, and employed single-molecule long-read sequencing (also known as Third-Generation Sequencing) of genomic DNA to analyze the genetic basis of CD36. The study identified 180 genetic variants, 12 of which were found to alter the amino acid sequence. Notably, four of these mutations (c.220 C > T; c.329_330delAC; c.430-1 G > C; c.1006 + 2 T > G) are premature termination mutations that lead to protein truncation. Using Fisher's exact test, we statistically analyzed a specific haplotype, c.-132A > C and c.329_330delAC, along with their clinical phenotypes, revealing a strong association between these variants in the 5' block and type I CD36 deficiency. We analyzed the CD36 gene sequences in platelet donors and patients with PTR (platelet transfusion refractoriness) and FNAIT (fetal and neonatal alloimmune thrombocytopenia), conducting a detailed haplotype analysis associated with type I CD36 deficiency and FNAIT.

Targeted long-read sequencing identifies missing pathogenic variant in unsolved 11ß-hydroxylase deficiency.

BACKGROUND: 11ß-hydroxylase deficiency (11ß-OHD), caused by homozygosity or compound heterozygosity CYP11B1 variants, is the second most common cause of congenital adrenal hyperplasia (CAH). Due to the high degree of sequence identity between CYP11B1 and CYP11B2, chimeric genes, and complex structural variants (SVs), the conventional approach to gene testing for 11ß-OHD is facing challenges. The study aimed to clarify the underlying genetic causes of two siblings of a Chinese family with 11ß-OHD. METHODS: Peripheral blood samples and clinical information were collected from subjects and their family members. Sex steroid concentrations were measured using LC-MS/MS. Long-range PCR-based next-generation sequencing (NGS), PCR assay and target long-read sequencing were used to detect the pathogenic variants. RESULTS: Early onset hypertension, increased serum levels of adrenocorticotropin (ACTH), progesterone, testosterone, and decreased cortisol and potassium were detected in both affected siblings. Long-range PCR-based NGS identified a heterozygous missense variant (NM_000497.4:c.281 C > T, p.P94> L) in CYP11B1 gene in the two siblings. PCR detected no chimeric CYP11B2/CYP11B1 gene. We finally identified a second pathogenic variant in CYP11B1 gene via target long-read sequencing (T-LRS). This novel variant was a deletion-insertion variant and located chr8:143957269-143,957,579 (hg19) with the insertion of 'ACAG' (NM_000497.4:c.954 + 78_980delinsACAG), which was in trans with CYP11B1: c.281 C > T. CONCLUSIONS: Our study suggests that the integrated long-range PCR-based NGS and T-LRS seem to be the most reliable and accurate method for 11ß-OHD genetic diagnosis and carrier sequencing.

Unravelling the complexity of bovine milk microbiome: insights into mastitis through enterotyping using full-length 16S-metabarcoding.

BACKGROUND: Mastitis, inflammation of the mammary gland, is a major disease of dairy cattle and the main cause for antimicrobial use. Although mainly caused by bacterial infections, the aetiological agent often remains unidentified by conventional microbiological culture methods. The aim of this study was to test whether shifts in the bovine mammary gland microbiota can result in initiation or progression of mastitis. METHODS: Oxford-Nanopore long-read sequencing was used to generate full-length 16S rRNA gene reads (16S-metabarcoding) to characterise the microbial population of milk from healthy and diseased udder of cows classified into five groups based on their mastitis history and parity. RESULTS: Samples were classified into six enterotypes, each characterised by a marker genus and several differentially-abundant genera. Two enterotypes were exclusively composed of clinical mastitis samples and displayed a marked dysbiosis, with a single pathogenic genus predominating and displacing the endogenous bacterial population. Other mastitis samples (all subclinical and half of the clinical) clustered with those from healthy animals into three enterotypes, probably reflecting intermediate states between health and disease. After an episode of clinical mastitis, clinical recovery and microbiome reconstitution do not always occur in parallel, indicating that the clinical definition of the udder health status does not consistently reflect the microbial profile. CONCLUSIONS: These results show that mastitis is a dynamic process in which the udder microbiota constantly changes, highlighting the complexity of defining a unique microbiota profile indicative of mastitis.

Insights into the length and breadth of methodologies harnessed to study human telomeres.

Telomeres are protective structures at the end of eukaryotic chromosomes that are strongly implicated in ageing and ill health. They attrition upon every cellular reproductive cycle. Evidence suggests that short telomeres trigger DNA damage responses that lead to cellular senescence. Accurate methods for measuring telomeres are required to fully investigate the roles that shortening telomeres play in the biology of disease and human ageing. The last two decades have brought forth several techniques that are used for measuring telomeres. This editorial highlights strengths and limitations of traditional and emerging techniques, guiding researchers to choose the most appropriate methodology for their research needs. These methods include Quantitative Polymerase Chain Reaction (qPCR), Omega qPCR (Ω-qPCR), Terminal Restriction Fragment analysis (TRF), Single Telomere Absolute-length Rapid (STAR) assays, Single TElomere Length Analysis (STELA), TElomere Shortest Length Assays (TESLA), Telomere Combing Assays (TCA), and Long-Read Telomere Sequencing. Challenges include replicating telomere measurement within and across cohorts, measuring the length of telomeres on individual chromosomes, and standardised reporting for publications. Areas of current and future focus have been highlighted, with recent methodical advancements, such as long-read sequencing, providing significant scope to study telomeres at an individual chromosome level.

Assembly of Mitochondrial Genomes Using Nanopore Long-Read Technology in Three Sea Chubs (Teleostei: Kyphosidae).

Complete mitochondrial genomes have become markers of choice to explore phylogenetic relationships at multiple taxonomic levels and they are often assembled using whole genome short-read sequencing. Herein, using three species of sea chubs as an example, we explored the accuracy of mitochondrial chromosomes assembled using Oxford Nanopore Technology (ONT) Kit 14 R10.4.1 long reads at different sequencing depths (high, low and very low or genome skimming) by comparing them to 'gold' standard reference mitochondrial genomes assembled using Illumina NovaSeq short reads. In two species of sea chubs, Girella nigricans and Kyphosus azureus, ONT long-read assembled mitochondrial genomes at high sequencing depths (> 25× whole [nuclear] genome) were identical to their respective short-read assembled mitochondrial genomes. Not a single 'homopolymer insertion', 'homopolymer deletion', 'simple substitution', 'single insertion', 'short insertion', 'single deletion' or 'short deletion' were detected in the long-read assembled mitochondrial genomes after aligning each one of them to their short-read counterparts. In turn, in a third species, Medialuna californiensis, a 25× sequencing depth long-read assembled mitochondrial genome was 14 nucleotides longer than its short-read counterpart. The difference in total length between the latter two assemblies was due to the presence of a short motif 14 bp long that was repeated (twice) in the long read but not in the short-read assembly. Read subsampling at a sequencing depth of 1× resulted in the assembly of partial or complete mitochondrial genomes with numerous errors, including, among others, simple indels, and indels at homopolymer regions. At 3× and 5× subsampling, genomes were identical (perfect) or almost identical (quasiperfect, 99.5% over 16,500 bp) to their respective Illumina assemblies. The newly assembled mitochondrial genomes exhibit identical gene composition and organisation compared with cofamilial species and a phylomitogenomic analysis based on translated protein-coding genes suggested that the family Kyphosidae is not monophyletic. The same analysis detected possible cases of misidentification of mitochondrial genomes deposited in GenBank. This study demonstrates that perfect (complete and fully accurate) or quasiperfect (complete but with a single or a very few errors) mitochondrial genomes can be assembled at high (> 25×) and low (3-5×) but not very low (1×, genome skimming) sequencing depths using ONT long reads and the latest ONT chemistries (Kit 14 and R10.4.1 flowcells with SUP basecalling). The newly assembled and annotated mitochondrial genomes can be used as a reference in environmental DNA studies focusing on bioprospecting and biomonitoring of these and other coastal species experiencing environmental insult. Given the small size of the sequencing device and low cost, we argue that ONT technology has the potential to improve access to high-throughput sequencing technologies in low- and moderate-income countries.

Novel Genes of the Male Reproductive System: Potential Roles in Male Reproduction and as Non-hormonal Male Contraceptive Targets.

The development of novel non-hormonal male contraceptives represents a pivotal frontier in reproductive health, driven by the need for safe, effective, and reversible contraceptive methods. This comprehensive review explores the genetic underpinnings of male fertility, emphasizing the crucial roles of specific genes and structural variants (SVs) identified through advanced sequencing technologies such as long-read sequencing (LRS). LRS has revolutionized the detection of structural variants and complex genomic regions, offering unprecedented precision and resolution over traditional next-generation sequencing (NGS). Key genetic targets, including those implicated in spermatogenesis and sperm motility, are highlighted, showcasing their potential as non-hormonal contraceptive targets. The review delves into the systematic identification and validation of male reproductive tract-specific genes, utilizing advanced transcriptomics and genomics studies with validation using novel knockout mouse models. We discuss the innovative application of small molecule inhibitors, developed through platforms like DNA-encoded chemistry technology (DEC-Tec), which have shown significant promise in preclinical models. Notable examples include inhibitors targeting serine/threonine kinase 33 (STK33), soluble adenylyl cyclase (sAC), cyclin-dependent kinase 2 (CDK2), and bromodomain testis associated (BRDT), each demonstrating nanomolar affinity and potential for reversible and specific inhibition of male fertility. This review also honors the contributions of Dr. David L. Garbers whose foundational work has paved the way for these advancements. The integration of genomic, proteomic, and chemical biology approaches, supported by interdisciplinary collaboration, is poised to transform male contraceptive development. Future perspectives emphasize the need for continued innovation and rigorous testing to bring these novel contraceptives from the laboratory to clinical application, promising a new era of male reproductive health management.

Identification of age-associated microbial changes via long-read 16S sequencing.

BACKGROUND: Age-related gut microbial changes have been widely investigated over the past decade. Most of the previous age-related microbiome studies were conducted on the Western population, and the short-read sequencing (e.g., 16S V4 or V3-V4 region) was the most common microbiota profiling method. We evaluated the gut compositional differences using the long-read sequencing approach (i.e., PacBio sequencing targeting the full-length V1-V9 regions) to enable a deeper taxonomic resolution and better characterize the gut microbiome of Singaporeans from different age groups. RESULTS: A total of 83 research participants were included in this study. Although no significant differences were detected in alpha and beta diversity, our study demonstrated several bacterial taxa with abundances that were significantly different across age groups. With young individuals as the reference group, Eggerthella lenta and Bacteroides uniformis were found to be significantly altered in the middle-aged group, while Catenibacterium mitsuokai and Bacteroides plebeius were significantly altered in the elderly group. These age-related differences in the gut microbiome were associated with aberrations in several predicted functional pathways, including dysregulations of pathways related to lipopolysaccharide and tricarboxylic acid cycle in older adults. CONCLUSIONS: The utilization of long-read sequencing facilitated the identification of species- and strain-level differences across age groups, which was challenging with the partial 16S rRNA sequencing approach. Nevertheless, replication studies are warranted to confirm our findings, and if confirmed, further in vitro and in vivo studies are crucial to better understand the impact of the altered levels of age-related bacterial taxa. Additionally, the modest performance of strain-level taxonomic classification using 16S-ITS-23S gene sequences, likely due to the limited depth of currently available alignment databases, highlights the need for optimization and refinement in curating these databases for the long-read sequencing approach.