Long-read Sequencing Research Articles

Improving Nanopore sequencing-based core genome MLST for global infection control: a strategy for GC-rich pathogens like Burkholderia pseudomallei.

This study highlights a significant advancement in genomic surveillance of bacterial pathogens, specifically addressing the challenges posed by the GC-rich species Burkholderia pseudomallei. Core genome multilocus sequence typing (cgMLST) is widely used for bacterial typing as it combines high resolution with simple implementation and standardization. To improve cost efficiency and thus accessibility, we changed the sequencing approach from Illumina short-read (SR) to Oxford Nanopore long-read sequencing (ONT-LR). ONT-LR-based cgMLST showed a very high error rate compared with SR-based cgMLST, most likely due to methylation-associated errors. PCR-based library preparation, which is proposed to correct these errors, did not achieve the required accuracy. In contrast, native barcoding with advanced basecalling and polishing strategies massively reduces allelic differences. This optimized ONT-LR cgMLST workflow provides a transformative solution for cost-efficient, high-resolution typing of B. pseudomallei. Furthermore, this study can serve as a guide for similarly challenging bacteria.

Computational Tools for Studying Genome Structural Variation.

Structural variation (SV) typically refers to alterations in DNA fragments at least 50 base pairs long in the human genome. It can alter thousands of DNA nucleotides and thus significantly influence human health, disease, and clinical phenotypes. There is a shared and growing recognition that the emergence of effective computational tools and high-throughput technologies such as short-read sequencing and long-read sequencing offers novel insight into SV and, by extension, diseases affecting planetary health. However, numerous available SV tools exist with varying strengths and weaknesses. This is currently hampering the abilities of scholars to select the optimal tools to study SVs. Here, we reviewed 175 tools developed in the past two decades for SV detection, annotation, visualization, and downstream analysis of human genomics. In this expert review, we provide a comprehensive catalog of SV-related tools across different technology platforms and summarize their features, strengths, and limitations with an eye to accelerate systems science and planetary health innovations.

Genome assembly of a living fossil, the Atlantic horseshoe crab Limulus polyphemus, reveals lineage-specific whole genome duplications, transposable element-based centromeres and a ZW sex chromosome system.

Horseshoe crabs, considered living fossils with a stable morphotype spanning ∼445 million years, are evolutionarily, ecologically and biomedically important species experiencing rapid population decline. Of the four extant species of horseshoe crabs, the Atlantic horseshoe crab, Limulus polyphemus, has become an essential component of the modern medicine toolkit. Here, we present the first chromosome-level genome assembly, and most contiguous and complete assembly to date, for Limulus polyphemus using nanopore long-read sequencing and chromatin conformation analysis. We find support for three horseshoe crab-specific whole genome duplications, but none shared with Arachnopulmonata (spiders and scorpions). Moreover, we discovered tandem duplicates of endotoxin-detection pathway components Factor C and Factor G, identify candidate centromeres consisting of Gypsy retroelements, and classify the ZW sex chromosome system for this species and a sister taxon, Carcinoscorpius rotundicauda. Finally, we revealed this species has been experiencing a steep population decline over the last 5 million years, highlighting the need for international conservation interventions and fisheries-based management for this critical species.

Full-length mRNA sequencing resolves novel variation in 5' UTR length for genes expressed during human CD4 T-cell activation.

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5' untranslated region (UTR). Differential 5' UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5' UTR lengthened by over 100bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.

Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency Detected by Long-Read Sequencing and Phenotypes Correlation.

21-Hydroxylase deficiency (21-OHD) is caused by pathogenic variants in CYP21A2. High homology between CYP21A2 and its pseudogene CYP21A1P causes mismatches, leading to deletions and CYP21A1P/CYP21A2 chimeras. To detect chimeric CYP21A1P/CYP21A2 in 21-OHD patients using long-read sequencing (LRS) and analyze genotype-phenotype correlations. From 2015 to 2023, 869 21-OHD patients were enrolled at Peking Union Medical College Hospital, with 113 identified harboring CYP21A2 large deletion. Long-range PCR and LRS were used to identify the types of CYP21A1P/CYP21A2 chimeric. Haplotype analysis explored founder effects, and in vitro assays assessed the functional impact of novel mutations. Clinical data were retrospectively collected and patients were classified into 4 groups based on genotypes and residual enzyme activity to study genotype-phenotype correlations. Ten types of chimeric CYP21A1P/CYP21A2 genes were identified across 119 alleles, including a novel type, CH-10. The most common, CH-1, accounted for 50.4% of all types. Haplotype analysis of 24 SNPs within CYP21A1P/CYP21A2 CH-1 revealed 25 haplotypes, with haplotype 11 being the most prevalent. Variants p.L100P and p.L301V of CYP21A2 showed enzyme activities of 1.36 ± 0.44% or 1.63 ± 0.19% for 17-hydroxyprogesterone to 11-deoxycortisol, and 1.36 ± 0.58% or 3.99 ± 1.09% for progesterone to 11-deoxycorticosterone, respectively, linked to the simple virilizing type. Genotype-phenotype consistency rates were 78.6% to 84% across the 4 groups. LRS is a comprehensive genetic testing method for 21-OHD patients, effectively detecting both CYP21A2 gene variants and CYP21A1P/CYP21A2 chimeric gene types. This study expands the CYP21A2 variant spectrum by identifying a novel chimera. Haplotype analysis revealed diverse haplotypes for each chimeric gene type, suggesting the absence of a common founder effect. The strong genotype-phenotype correlation aids genetic counseling and supports personalized treatment.

A Near Complete Genome Assembly of the Oshima Cherry Cerasus speciosa

The Oshima cherry (Cerasus speciosa), which is endemic to Japan, has significant cultural and horticultural value. In this study, we present a near complete telomere-to-telomere genome assembly for C. speciosa, derived from the old growth “Sakurakkabu” tree on Izu Oshima Island. Using Illumina short-read, PacBio long-read, and Hi-C sequencing, we constructed a 269.3 Mbp genome assembly with a contig N50 of 32.0 Mbp. We examined the distribution of repetitive sequences in the assembled genome and identified regions that appeared to be centromeric. Detailed structural analysis of these putative centromeric regions revealed that the centromeric regions of C. speciosa comprised repetitive sequences with monomer lengths of 166 or 167 bp. Comparative genomic analysis with Prunus sensu lato genome revealed structural variations and conserved syntenic regions. This high-quality reference genome provides a crucial tool for studying the genetic diversity and evolutionary history of Cerasus species, facilitating advancements in horticultural research and the preservation of this iconic species.

Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples

Since viruses are obligate intracellular pathogens, sequencing their genomes results in metagenomic data from both the virus and the host. Virology researchers are constantly seeking new, cost-effective strategies and bioinformatic pipelines for the retrieval of complete viral genomes from these metagenomic samples. Avian orthoreoviruses (ARVs) pose a significant and growing threat to the poultry industry and frequently cause economic losses associated with disease in production birds. Currently available commercial vaccines are ineffective against new ARV variants and ARV outbreaks are increasing worldwide, requiring whole genome sequencing (WGS) to characterize strains that evade vaccines. This study compares the effectiveness of long-read and short-read sequencing technologies for obtaining ARV complete genomes. We used eight clinical isolates of ARV, each previously processed using our published viral genome enrichment protocol. Additionally, we evaluate three assembly methods to determine which provided the most complete and reliable whole genomes: De novo, reference-guided or hybrid. The results suggest that our ARV genome enrichment protocol caused some fragmentation of the viral cDNA that impacted the length of the long reads (but not the short reads) and, as a result, caused a failure to produce complete genomes via de novo assembly. Overall, we observed that regardless of the sequencing technology, the best quality assemblies were generated by mapping quality-trimmed reads to a custom reference genome. The custom reference genomes were in turn constructed with the publicly available ARV genomic segments that shared the highest sequence similarity with the contigs from short-read de novo assemblies. Hence, we conclude that short-read sequencing is the most suitable technology to combine with our ARV genome enrichment protocol.

Fraxinellone-mediated targeting of cathepsin B leakage from lysosomes induces ferroptosis in fibroblasts to inhibit hypertrophic scar formation

BackgroundHypertrophic scar (HS) is a common fibrotic skin disorder characterized by the excessive deposition of extracellular matrix (ECM). Fibroblasts are the most important effector cells involved in HS formation. Currently no satisfactory treatment has been developed.MethodsThe impact of fraxinellone (FRA) on the proliferation and migration capacity of human hypertrophic scar-derived fibroblasts (HSFs) was assessed by EdU proliferation, wound healing and transwell assays. Quantitative real-time PCR (qRT‒PCR), Western blot (WB), immunofluorescence staining and collagen gel contraction assays were performed to evaluate the collagen production and activation capacity of HSFs. Oxford Nanopore Technologies long-read RNA sequencing (ONT long-read RNA-seq) revealed the occurrence of ferroptosis in HSF and ferroptosis executioner-cathepsin B (CTSB). The mechanisms underlying FRA-induced HSF ferroptosis were examined through fluorescence staining, qRT‒PCR, WB and molecular docking study. The therapeutic efficacy of FRA was further validated in vivo using a rabbit ear scar model.ResultsFRA treatment significantly suppressed the proliferation, migration, collagen production and activation capacity of HSFs. ONT long-read RNA-seq discovered that FRA modulated the expression of transcripts related to ferroptosis and lysosomes. Mechanistically, FRA treatment reduced the protein expression level of glutathione peroxidase 4 (GPX4) and induced the release of CTSB from lysosomes into the cytoplasm. CTSB further induced ferroptosis via spermidine/spermine-N1-acetyltransferase (SAT1)-mediated lipid peroxidation, mitochondrial damage and mitogen-activated protein kinase (MAPK) signalling pathway activation, eventually affecting the function of HSFs. Moreover, FRA treatment attenuated the formation of HS in rabbit ears via CTSB-mediated ferroptosis. The antifibrotic effects of FRA were abrogated by pretreatment with a CTSB inhibitor (CA-074-me).ConclusionsThis study reveals that FRA ameliorates HS by inducing CTSB leakage from lysosomes, causing SAT1-mediated lipid peroxidation, mitochondrial damage and MAPK signalling pathway activation, thus mediating HSF ferroptosis. Therefore, FRA could be a promising therapeutic agent for treating HS.

The additional diagnostic yield of long-read sequencing in undiagnosed rare diseases

Long-read sequencing (LRS) is a promising technology positioned to study the significant proportion of rare diseases (RDs) that remain undiagnosed as it addresses many of the limitations of short-read sequencing, detecting and clarifying additional disease-associated variants that may be missed by the current standard diagnostic workflow for RDs. Some key areas where additional diagnostic yields may be realized include: (1) detection and resolution of structural variants (SVs); (2) detection and characterization of tandem repeat expansions; (3) coverage of regions of high sequence similarity; (4) variant phasing; (5) the use of de novo genome assemblies for reference-based or graph genome variant detection; and (6) epigenetic and transcriptomic evaluations. Examples from over 50 studies support that the main areas of added diagnostic yield currently lie in SV detection and characterization, repeat expansion assessment, and phasing (with or without DNA methylation information). Several emerging studies applying LRS in cohorts of undiagnosed RDs also demonstrate that LRS can boost diagnostic yields following negative standard-of-care clinical testing and provide an added yield of 7%–17% following negative short-read genome sequencing. With this evidence of improved diagnostic yield, we discuss the incorporation of LRS into the diagnostic care pathway for undiagnosed RDs, including current challenges and considerations, with the ultimate goal of ending the diagnostic odyssey for countless individuals with RDs.

Transcriptomic Analysis Reveals Patterns of Expression of Stage-Specific Genes in Early Apis cerana Embryos

Background/Objectives: Apis cerana development is described as comprising four stages: embryo, larva, pupa, and adult. There are significant differences between workers and drones in terms of physiological functions and social roles, and the formation of the organ primordia occurs during the embryonic stage. Therefore, the objective of this study is to investigate the differential expression of and alternative splicing of genes in worker and drone embryos and to explain their unique developmental patterns. Methods: Long-read sequencing (PacBio Iso-Seq) and short-read sequencing (Illumina RNA-Seq) were used to investigate worker and drone embryo gene expression differences in A. cerana across five developmental points (12, 24, 36, 48, and 60 h). Results: The study identified 59,254 common isoforms, with 5744 and 5106 isoforms specific to worker and drone embryos, respectively. Additionally, a new transcript of the csd gene was identified. The number of differentially expressed genes (3391) and differential splicing events (470 genes) peaked at the 24-h embryonic stage. Differential splicing events of csd, dsx, and Y-y were observed in the worker and drone embryos. Conclusions: The gene expression results indicated that the 24-h embryonic point is a critical period for the expression of genes related to developmental and behavioral differences between workers and drones. The findings provide a theoretical basis for future research on the developmental differences between workers and drones.

Enhancing diagnostic outcomes in kidney genetic disorders: the KidGen national kidney genomics study protocol

BackgroundGenetic kidney disease (GKD) significantly affects the community and is responsible for a notable portion of adult kidney disease cases and about half of cases in paediatric patients. It substantially impacts the quality of life and life expectancy for affected children and adults across all stages of kidney disease. Precise genetic diagnosis in GKD promises to improve patient outcomes, provide access to targeted treatments, and reduce the disease burden for individuals, families, and healthcare systems. Genetic investigations are increasingly used in nephrology practice; however, many patients who undergo testing still lack a definitive diagnosis.MethodsThe KidGen National Kidney Genomics Study aims to increase diagnostic yield for those with suspected monogenic kidney disease without a diagnosis after standard diagnostic genetic testing. The program will seek to enrol up to 200 families from KidGen Collaborative kidney genetics clinics across Australia who have yet to receive conclusive diagnoses despite prior testing. Participants will undergo a personalised pathway of research genomic investigations. These include re-analysing existing data and/or undergoing advanced genomic testing methods, including short and long-read whole-genome sequencing, RNA sequencing, and functional genomics strategies using mouse modelling or kidney organoids.DiscussionThe KidGen National Kidney Genomics Study is a coordinated, multidisciplinary extension of previous research projects that aims to assess the diagnostic yield of advanced genomic approaches. The study's evidence will drive changes to current diagnostic pathways, including identifying which chronic kidney disease patients are most likely to benefit from a more comprehensive genomic approach to diagnosis.

Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Double carbapenemases in Klebsiella pneumoniae blood isolates: dissemination in a single medical center via multiple plasmids and a variety of highly efficient clones.

Acquisition of multiple carbapenemase genes by Klebsiella pneumoniae (Kp) is an emerging public health threat. Here, we aim to elucidate the population structure of Kp blood isolates carrying two different carbapenemase genes and identify the mechanism facilitating their dissemination. The study was conducted in a tertiary healthcare center between 2014 and 2022. Twenty-four patients with bacteremia caused by Kp carrying two different carbapenemase genes were identified. All 24 blood isolates were analyzed by short-read genome sequences supplemented by long reads in a selected number of isolates. All isolates carried blaKPC (23 blaKPC-2, 1 blaKPC-3) and blaVIM-1 genes, along with a variety of antimicrobial resistance determinants. The isolates were clustered in six clonal lineages (ST39, ST147, ST323, ST258, ST3035, and ST340). Long-read genome sequences demonstrated that each carbapenemase gene was located in a separate group of plasmids: the blaKPC-2 on a fusion of IncFIB(pQil) and IncFII(K) plasmids, the blaKPC-3 on IncX3, the blaVIM-1 on IncC, or a fusion of the IncFIB(pNDM-Mar) and IncHI1B(pNDM-MAR) plasmids. Comparison of plasmid content of eight isolates carrying a single carbapenemase gene from a previous study with eight isolates carrying two carbapenemase genes from the present study, matched by clonal lineages, revealed that the second carbapenemase gene was acquired by addition of another plasmid. Identical plasmids were found within the same lineage and across lineages. These findings suggest that dissemination of carbapenemase genes in our hospital setting was driven by multiple plasmids across a variety of highly efficient clones.

Long-read 16S rRNA amplicon sequencing reveals microbial characteristics in patients with colorectal adenomas and carcinoma lesions in Egypt

BackgroundColorectal cancer (CRC) is among the five leading causes of cancer incidence and mortality. During the past decade, the role of the gut microbiota and its dysbiosis in colorectal tumorigenesis has been emphasized. Metagenomics and amplicon-based microbiome profiling provided insights into the potential role of microbial dysbiosis in the development of CRC.AimTo address the scarcity of information on differential microbiome composition of tumor tissue in comparison to adenomas and the lack of such data from Egyptian patients with CRC.Materials and methodsLong-read nanopore sequencing of 16S rRNA amplicons was used to profile the colonic microbiota from fresh colonoscopic biopsy samples of Egyptian patients with CRC and patients with colonic polyps.ResultsSpecies richness of CRC lesions was significantly higher than that in colonic polyps (p-value = 0.0078), while evenness of the CRC group was significantly lower than the colonic polyps group (p-value = 0.0055). Both species richness and Shannon diversity index of the late onset CRC samples were significantly higher than those of the early onset ones. The Firmicutes-to-Bacteroidetes (F/B) ratio was significantly higher in the CRC group than in the colonic polyps group (p-value = 0.0054), and significantly higher in samples from early-onset CRC. The Enterococcus spp. were significantly overabundant in patients with rectal cancer and early-onset CRC, while Staphylococcus spp. were significantly higher in patients with sigmoid cancer and late-onset CRC. In addition, the relative abundance of Fusobacterium nucleatum was significantly higher in CRC patients.ConclusionDifferentiating trends were identified at phylum, genus, and species levels, despite the inter-individual differences. In summary, this study addressed the microbial dysbiosis associated with CRC and colonic polyps groups, paving the way for a better understanding of the pathogenesis of early and late-onset CRC in Egyptian patients.

Acute Vestibular Syndrome Unmasking an RFC1-Spectrum Disorder.

Since the discovery of biallelic pentanucleotide expansions in RFC1 as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome, a wide and growing clinical spectrum has emerged. In this article, we report a man with acute vestibular syndrome that likely unmasked a RFC1-spectrum disorder. Detailed clinical evaluation, neuroimaging, nerve conduction studies, evaluation of vestibular function, and short-read whole-genome sequencing and targeted long-read adaptive sequencing were performed. Clinical follow-up after acute vestibular syndrome revealed bilateral vestibular areflexia and a gait abnormality with the Scale for the Assessment and Rating of Ataxia score of 5. Brain MRI was normal while 2 electroneurography tests did not show neuropathy. However, severe cough spells raised the suspicion of a RFC1-spectrum disorder. WGS screening detected a recessive intronic pentanucleotide expansion in RFC1, which was verified and sized using long-read adaptive sequencing. This is an unusual presentation; oscillopsia after an acute vestibular syndrome and cough spells should alert clinicians about a RFC1-spectrum disorder, even in the absence of neuropathy and neuroradiologic abnormalities.

Overview of the etiology of childhood cancer and future directions.

We provide an overview of the etiology of childhood cancer, the state of the literature, and highlight some opportunities for future research, including technological advancements that could be applied to etiologic studies of childhood cancer to accelerate our understanding. Risk factors of childhood cancer were summarized based on demographics and perinatal factors, environmental risk factors, and genetic risk factors. Overall, demographics and perinatal factors are the most well studied in relation to childhood cancer. While environmental risk factors have been implicated, more work is needed to pinpoint specific exposures, identify window(s) of susceptibility, and understand mechanisms. With genome-wide association studies (GWAS), genetic risk factors of eight childhood cancers have emerged, and opportunities remain to conduct GWAS for other cancer types and determine whether risk variants are inherited or de novo. Technological advancements that can shed light into the susceptibility of childhood cancer include metabolomics, using primary teeth as an exposure matrix, and long-read sequencing. The development of childhood cancer remains largely not well understood. Collaboration to increase sample size to conduct analyses by histology and/or molecular subtype and application of novel technologies will accelerate our understanding of childhood cancer.

Detection and Whole-Genome Analysis of tigecycline resistant Escherichia coli in poultry and meat samples in Türkiye.

The emergence and dissemination of tigecyline resistant Enterobacterales (TRE) in animals is a critical issue. This study aimed to investigate the presence of TRE in the gut of healthy avians as well as meat samples. A total of 940 ceacal samples from 94 commercial poultry flocks were collected at slaughter and a total of 335 meat samples [(chicken (n = 159), turkey (n = 4) and beef/lamb (n = 172)] were collected from supermarkets and butcher shops. Out of 960 samples, 146 (19.21 %) samples from chicken farms and 24 (13.3 %) from turkey farms were positive for TRE. Forty-nine Escherichia coli isolates were determined to carry the tet(X4) gene by PCR and exhibited multi-drug resistance. Whole-genome short-read sequencing (WGS) on all tet(X4) positive E. coli isolates and long-read sequencing on a selection of five isolates were carried out. WGS identified four ST types (ST206 being the most dominant, ST609, ST744 and ST189), indicating significant homogeneity among tigecyline resistant E. coli strains. In 47 isolates, the tet(X4) gene was transferrable to E. coli EC600 and it was found to be located on the IncX1 plasmid. Additionally, all tet(X4)-positive E. coli isolates also harbored other resistance genes, including floR, aadA2 and tet(A). In this study, the identification of tet(X4) carrying E. coli in healthy chicken and meats suggests the likely source of food-producing animals for humans. Therefore, active surveillance of critical priority lineages of TRE should focus on to contain the potential public health risk.

Toward a comprehensive profiling of alternative splicing proteoform structures, interactions and functions.

The mRNA splicing machinery has been estimated to generate 100,000 known protein-coding transcripts for 20,000 human genes (Ensembl, Sept. 2024). However, this set is expanding with the massive and rapidly growing data coming from high-throughput technologies, particularly single-cell and long-read sequencing. Yet, the implications of splicing complexity at the protein level remain largely uncharted. In this review, we describe the current advances toward systematically assessing the contribution of alternative splicing to proteome function diversification. We discuss the potential and challenges of using artificial intelligence-based techniques in identifying alternative splicing proteoforms and characterising their structures, interactions, and functions.

Perspectives and opportunities in forensic human, animal, and plant integrative genomics in the Pangenome era.

The Human Pangenome Reference Consortium, the Chinese Pangenome Consortium, and other plant and animal pangenome projects have announced the completion of pilot work aimed at constructing high-quality, haplotype-resolved reference graph genomes representative of global ethno-linguistically different populations or different plant and animal species. These graph-based, gapless pangenome references, which are enriched in terms of genomic diversity, completeness, and contiguity, have the potential for enhancing long-read sequencing (LRS)-based genomic research, as well as improving mappability and variant genotyping on traditional short-read sequencing platforms. We comprehensively discuss the advancements in pangenome-based genomic integrative genomic discoveries across forensic-related species (humans, animals, and plants) and summarize their applications in variant identification and forensic genomics, epigenetics, transcriptomics, and microbiome research. Recent developments in multiplexed array sequencing have introduced a highly efficient and programmable technique to overcome the limitations of short forensic marker lengths in LRS platforms. This technique enables the concatenation of short RNA transcripts and DNA fragments into LRS-optimal molecules for sequencing, assembly, and genotyping. The integration of new pangenome reference coordinates and corresponding computational algorithms will benefit forensic integrative genomics by facilitating new marker identification, accurate genotyping, high-resolution panel development, and the updating of statistical algorithms. This review highlights the necessity of integrating LRS-based platforms, pangenome-based study designs, and graph-based pangenome references in short-read mapping and LRS-based innovations to achieve precision forensic science.

Superior Resolution Profiling of the Coleofasciculus Microbiome by Amplicon Sequencing of the Complete 16S rRNA Gene and ITS Region.

The filamentous cyanobacterium Coleofasciculus chthonoplastes is the key primary producer of marine microbial mats. We elucidated the microbiomes of 32 non-axenic Coleofasciculus isolates using PacBio-based amplicon sequencing of the complete 16S rRNA gene and the internally transcribed spacer (16S-ITS). The length of authentic amplicon sequence variants (ASVs) ranged from 1827 to 3044 nucleotides (median: 2267 nt). The results, which were complemented by metagenome analyses and cultivation approaches, revealed the presence of more than 70 associated heterotrophs in the culture of Coleofasciculus sp. WW12. The great bacterial diversity in the cyanosphere is dominated by Pseudomonadota (59%) and Bacteroidota (23%). Allelic ribosomal operon variants were detected in 18 Coleofasciculus strains and our analyses proposed the presence of at least four different species. A comparative analysis of cyanobacterial microbiomes documented complementary advantages of amplicon sequencing versus metagenomics with an individual strength of the 16S-ITS approach in terms of (i) ribosomal target sequence quality, (ii) contaminant detection and (iii) identification of rare bacteria. The characterisation of the Coleofasciculus microbiome showed that long-read amplicon sequencing of the 16S-ITS region is the method of choice for rapid profiling of non-axenic cyanobacteria. Its superior resolution allows a reliable differentiation of even very closely related strains.