Massively Parallel Sequencing Research Articles

TARGETED SEQUENCING REVEAL TWO PATHOGENIC MUTATIONS IN LDLR AND APOE GENES IN PATIENT WITH FAMILY HYPERCHOLESTEROLEMIA: CASE REPORT

Familial hypercholesterolemia (FH) is a serious inherited disorder that can lead to early development of cardiovascular disease (CVD) due to the high blood cholesterol levels. In our study, we identified in FH patient likely pathogenic variant of LDLR and APOE genes. Interest in the homozygous state of FH is understandable given its rarity and severe health consequences. The homozygous state, when both LDL receptor alleles mutated, demonstrates even higher cholesterol levels and an earlier and more severe clinical course compared with the heterozygous state. In our study, we report an interesting case of homozygous FH due to its rare prevalence. Patient U., 16 years old, woman, admitted to the National Research Cardiac Surgery Center (NRCC), was diagnosed with FH, congenital heart disease (CHD), bicuspidal aortic valve (AV) type 1. Both parents (mother and father) were included for reliable analysis. DNA was isolated from the venous blood sample by using the column method of QIAamp DNA Mini Kit. Targeted NGS using TruSight Cardio panel for 174 genes was performed on MiSeq, Illumina. Identified genetic variants were classified according to the ACMG guidelines. Pathogenic variants were validated by Sanger sequencing. Additionally, databases of the VarSome and ClinVar were used for analysis. Sequencing of 96 genes and bioinformatics analysis identified 2 likely pathogenic variants - C526T:p.R176C in APOE and G295A:p.E99K in LDLR gene, 4 uncertain significance variants in APOB, TTN, COL3A1, NOTCH1 and 90 benign variants in APOB, TTN, LAMA4, MYPN, DMD. LDLR (G295A:p.E99K) and APOE (C526T:p.R176C) were validated and confirmed by Sanger sequencing in patients and her father and mother. The main function of the APOE gene is to make the protein apolipoprotein E, which is involved in fat metabolism. In our study, the patient had a heterozygous variant of СT polymorphism rs7412 in the APOE gene, which indicates the presence of a possible disorder of fat metabolism. LDLR gene encodes a protein called low-density lipoprotein receptor, which is responsible for the uptake of cholesterol-carrying lipoprotein particles in cells. In our study, a patient with a homozygous AA variant of the LDLR gene was found to be pathogenic. Disruption of the uptake of cholesterol-carrying lipoprotein particles in cells can have serious consequences for the body. This can lead to cholesterol accumulation in the blood and tissues, which in turn can contribute to the development of atherosclerosis and other CVD. We found that both parents had a heterozygous genotype of G295A:p.E99K in LDLR gene; and mother had a heterozygous genotype and father had a homozygous genotype of C526T:p.R176C in APOE gene. In our study, we found the genetic mutations - two likely pathogenic genetic variants LDLR (G295A:p.E99K) and APOE (C526T:p.R176C) associated with FH in patient with clinical phenotype of FH using TruSight Cardio targeted panel of 96 genes. Genetic testing using targeted NGS or whole exome sequencing would be useful for right diagnosis and might improve personalize treatment for these patients. To identify other genes associated with FH whole exome sequencing can be recommend. Supported by Committee of Science of the Ministry of Science and Higher Education of Republic of Kazakhstan (AP14869903), (BR21881970) and Nazarbayev University CRP (211123CRP1608).

Certainty in uncertainty: Determining the rate and reasons for reclassification of variants of uncertain significance in haematological malignancies

AbstractIntroductionVariants of uncertain significance (VUS) are commonly reported in cancer with the widespread adoption of diagnostic massive parallel sequencing. The rate of reclassification of VUS in patients with haematological malignancy is not known and we evaluated this retrospectively. We also investigated whether re‐evaluating VUS in 12–24 months or greater than 24 months post‐initial classification was significant.MethodA retrospective audit of patients with haematological malignancies referred to the Molecular Medicine Department at the John Hunter Hospital in Newcastle, Australia between September 2018 and December 2021. Data was analysed for VUS, which was then re‐analysed in standard software using current somatic variant guidelines. Proportions of VUS at baseline were compared to post‐re‐analysis.ResultsThe most common diagnoses in the patient cohort (n = 944) were acute myelogenous leukaemia (41%), myelodysplastic syndrome (31%), and chronic myelomonocytic leukaemia (7%). A total of 210 VUS were re‐analysed. The most common VUS were in the TET2 (20%), RUNX1 (10%) and DNMT3A (9%) genes. A total of 103 were re‐analysed at 24–39 months post‐initial classification and 107 variants were re‐analysed between 12 and 24 months post‐initial classification. Of these, 33 (16%) of VUS were re‐classified at 24–39 months and 12 (11%) were re‐classified at 12–24 months post‐initial classification. The most common variants that were re‐classified in both groups were CSF3R (32%), TET2 (29%), ASXL1 (11%) and ZRSR2 (11%).ConclusionThis study on reclassification of VUS in blood cancers demonstrated that one in seven VUS were re‐classified 12 months post initial classification. This can inform practice guidelines and potentially impact the prognosis, diagnosis and treatment of haematological malignancies.

When is an SNP not an SNP?

Genomic duplications are important sources of structural change and gene innovation. In humans, the most recent and highly identical sequences (>90% homology, >1 kb long) are known as segmental duplications (SDs). Single-nucleotide variants or single-nucleotide polymorphisms within SDs have not been systematically assessed due to limitations around mapping short-read sequencing data. Single-nucleotide variant rs62486260 was flagged in a study of familial renal stone disease but it was unclear whether it was real or an artifact resulting from the presence of a SD. We describe in silico and wet-lab approaches to investigate this, using segment-specific long-PCR assays, followed by short PCR for Sanger sequencing. Our conclusion was that rs62486260 is an artifact. Our approach can be generalized to deal with other such situations.

Molecular age prediction using skull bone samples from individuals with and without signs of decomposition: a multivariate approach combining analysis of posttranslational protein modifications and DNA methylation.

The prediction of the chronological age of a deceased individual at time of death can provide important information in case of unidentified bodies. The methodological possibilities in these cases depend on the availability of tissues, whereby bones are preserved for a long time due to their mineralization under normal environmental conditions. Age-dependent changes in DNA methylation (DNAm) as well as the accumulation of pentosidine (Pen) and D-aspartic acid (D-Asp) could be useful molecular markers for age prediction. A combination of such molecular clocks into one age prediction model seems favorable to minimize inter- and intra-individual variation. We therefore developed (I) age prediction models based on the three molecular clocks, (II) examined the improvement of age prediction by combination, and (III) investigated if samples with signs of decomposition can also be examined using these three molecular clocks. Skull bone from deceased individuals was collected to obtain a training dataset (n = 86), and two independent test sets (without signs of decomposition: n = 44, with signs of decomposition: n = 48). DNAm of 6 CpG sites in ELOVL2, KLF14, PDE4C, RPA2, TRIM59 and ZYG11A was analyzed using massive parallel sequencing (MPS). The D-Asp and Pen contents were analyzed by high performance liquid chromatography (HPLC). Age prediction models based on ridge regression were developed resulting in mean absolute errors (MAEs)/root mean square errors (RMSE) of 5.5years /6.6 years (DNAm), 7.7 years /9.3 years (Pen) and 11.7 years /14.6 years (D-Asp) in the test set. Unsurprisingly, a general lower accuracy for the DNAm, D-Asp, and Pen models was observed in samples from decomposed bodies (MAE: 7.4-11.8 years, RMSE: 10.4-15.4 years). This reduced accuracy could be caused by multiple factors with different impact on each molecular clock. To acknowledge general changes due to decomposition, a pilot model for a possible age prediction based on the decomposed samples as training set improved the accuracy evaluated by leave-one-out-cross validation (MAE: 6.6-12 years, RMSE: 8.1-15.9 years). The combination of all three molecular age clocks did reveal comparable MAE and RMSE results to the pure analysis of the DNA methylation for the test set without signs of decomposition. However, an improvement by the combination of all three clocks was possible for the decomposed samples, reducing especially the deviation in case of outliers in samples with very high decomposition and low DNA content. The results demonstrate the general potential in a combined analysis of different molecular clocks in specific cases.

Bridging the gap between molecular and genomic epidemiology in tuberculosis: inferring MIRU-VNTR patterns from genomic data.

The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology.

Genomic insights into the antimicrobial resistance and virulence of Helicobacter pylori isolates from gastritis patients in Pereira, Colombia

BackgroundHelicobacter pylori infects the stomach and/or small intestines in more than half of the human population. Infection with H. pylori is the most common cause of chronic gastritis, which can lead to more severe gastroduodenal pathologies such as peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. H. pylori infection is particularly concerning in Colombia in South America, where > 80% of the population is estimated to be infected with H. pylori and the rate of stomach cancer is one of the highest in the continent.ResultsWe compared the antimicrobial susceptibility profiles and short-read genome sequences of five H. pylori isolates obtained from patients diagnosed with gastritis of varying severity (chronic gastritis, antral erosive gastritis, superficial gastritis) in Pereira, Colombia sampled in 2015. Antimicrobial susceptibility tests revealed the isolates to be resistant to at least one of the five antimicrobials tested: four isolates were resistant to metronidazole, two to clarithromycin, two to levofloxacin, and one to rifampin. All isolates were susceptible to tetracycline and amoxicillin. Comparative genome analyses revealed the presence of genes associated with efflux pump, restriction modification systems, phages and insertion sequences, and virulence genes including the cytotoxin genes cagA and vacA. The five genomes represent three novel sequence types. In the context of the Colombian and global populations, the five H. pylori isolates from Pereira were phylogenetically distant to each other but were closely related to other lineages circulating in the country.ConclusionsH. pylori from gastritis of different severity varied in their antimicrobial susceptibility profiles and genome content. This knowledge will be useful in implementing appropriate eradication treatment regimens for specific types of gastritis. Understanding the genetic and phenotypic heterogeneity in H. pylori across the geographical landscape is critical in informing health policies for effective disease prevention and management that is most effective at local and country-wide scales. This is especially important in Colombia and other South American countries that are poorly represented in global genomic surveillance studies of bacterial pathogens.

Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses

BackgroundMetagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity.MethodsWe evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers.ResultsCapture with the Twist CVRP increased sensitivity by at least 10–100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600–6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT.ConclusionsMetagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.

Integrative analyses of long and short-read RNA sequencing reveal the spliced isoform regulatory network of seedling growth dynamics in upland cotton.

The polyploid genome of cotton has significantly increased the transcript complexity. Recent advances in full-length transcript sequencing are now widely used to characterize the complete landscape of transcriptional events. Such studies in cotton can help us to explore the genetic mechanisms of the cotton seedling growth. Through long-read single-molecule RNA sequencing, this study compared the transcriptomes of three yield contrasting genotypes of upland cotton. Our analysis identified different numbers of spliced isoforms from 31,166, 28,716, and 28,713 genes in SJ48, Z98, and DT8 cotton genotypes, respectively, most of which were novel compared to previous cotton reference transcriptomes, and showed significant differences in the number of exon structures and coding sequence length due to intron retention. Quantification of isoform expression revealed significant differences in expression in the root and leaf of each genotype. An array of key isoform target genes showed protein kinase or phosphorylation functions, and their protein interaction network contained most of the circadian oscillator proteins. Spliced isoforms from the GIGANTEA (GI) protien were differentially regulated in each genotype and might be expected to regulate translational activities, including the sequence and function of target proteins. In addition, these spliced isoforms generate diurnal expression profiles in cotton leaves, which may alter the transcriptional regulatory network of seedling growth. Silencing of the novel spliced GI isoform Gh_A02G0645_N17 significantly affected biomass traits, contributed to variable growth, and increased transcription of the early flowering pathway gene ELF in cotton. Our high-throughput hybrid sequencing results will be useful to dissect functional differences among spliced isoforms in the polyploid cotton genome.

The first Chromosomal-level genome assembly of Sageretia thea using Nanopore long reads and Pore-C technology

Sageretia thea, a notable species within the mock buckthorn genus, is recognized for its intriguing biogeographical distribution and diverse medicinal properties. Despite this significance, genomic studies on S. thea are still in the nascent stages. We present the first chromosome-level genome assembly of S. thea that was generated using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies complemented by Pore-C chromatin conformation capture. The genome assembly had a size of 197.8 Mb with 12 chromosomal scaffolds and a scaffold N50 length of 15.9 Mb. A total of 25,434 protein-coding genes were identified and functionally annotated, and the gene model indicated 96.5% complete eukaryotic BUSCOs. Additionally, orthologous gene profiling and synteny analysis were performed to elucidate the evolutionary relationships within the Rhamnaceae family and Rosales. This high-quality chromosomal genome is the first genomic view of S. thea, which will serve as the basis for future studies on its biological and medicinal properties, and evolutionary history.

Single-molecule sequencing of the whole HCV genome revealed envelope deletions in decompensated cirrhosis associated with NS2 and NS5A mutations.

Defective hepatitis C virus (HCV) genomes with deletion of the envelope region have been occasionally reported by short-read sequencing analyses. However, the clinical and virological details of such deletion HCV have not been fully elucidated. We developed a highly accurate single-molecule sequencing system for full-length HCV genes by combining the third-generation nanopore sequencing with rolling circle amplification (RCA) and investigated the characteristics of deletion HCV through the analysis of 21 patients chronically infected with genotype-1b HCV. In 5 of the 21 patients, a defective HCV genome with approximately 2000bp deletion from the E1 to NS2 region was detected, with the read frequencies of 34-77%, suggesting the trans-complementation of the co-infecting complete HCV. Deletion HCV was found exclusively in decompensated cirrhosis (5/12 patients), and no deletion HCV was observed in nine compensated patients. Comparing the amino acid substitutions between the deletion and complete HCV (DAS, deletion-associated substitutions), the deletion HCV showed higher amino acid mutations in the ISDR (interferon sensitivity-determining region) in NS5A, and also in the TMS (transmembrane segment) 3 to H (helix) 2 region of NS2. Defective HCV genome with deletion of envelope genes is associated with decompensated cirrhosis. The deletion HCV seems susceptible to innate immunity, such as endogenous interferon with NS5A mutations, escaping from acquired immunity with deletion of envelope proteins with potential modulation of replication capabilities with NS2 mutations. The relationship between these mutations and liver damage caused by HCV deletion is worth investigating.

Infectious bronchitis virus vaccination, but not the presence of XCR1, is correlated with large differences in chicken caecal microbiota.

The chicken immune system and microbiota play vital roles in maintaining gut homeostasis and protecting against pathogens. In mammals, XCR1+ conventional dendritic cells (cDCs) are located in the gut-draining lymph nodes and play a major role in gut homeostasis. These cDCs sample antigens in the gut luminal contents and limit the inflammatory response to gut commensal microbes by generating appropriate regulatory and effector T-cell responses. We hypothesized that these cells play similar roles in sustaining gut homeostasis in chickens, and that chickens lacking XCR1 were likely to contain a dysbiotic caecal microbiota. Here we compare the caecal microbiota of chickens that were either heterozygous or homozygous XCR1 knockouts, that had or had not been vaccinated for infectious bronchitis virus (IBV). We used short-read (Illumina) and long-read (PacBio HiFi) metagenomic sequencing to reconstruct 670 high-quality, strain-level metagenome assembled genomes. We found no significant differences between alpha diversity or the abundance of specific microbial taxa between genotypes. However, IBV vaccination was found to correlate with significant differences in the richness and beta diversity of the microbiota, and to the abundance of 40 bacterial genera. In conclusion, we found that a lack of XCR1 was not correlated with significant changes in the chicken microbiota, but IBV vaccination was.

TRICHINELLA MURRELLI POZIO AND LA ROSA, 2000 IN A GRAY FOX (UROCYON CINEREOARGENTEUS) FROM PENNSYLVANIA: A NEW HOST RECORD FOR THIS ZOONOTIC NEMATODE.

Trichinella murrelli Pozio and La Rosa, 2000, is the primary sylvatic trichinellid encountered in temperate North America. During a survey for Sarcocystis in wild canids, a single worm matching the morphology of encapsulated Trichinella was observed in a muscle tissue squash from a gray fox male originating from Pennsylvania. The worm was photographed and then separated from the host tissue by artificial digestion, and genomic DNA was extracted from the worm. This DNA was subjected to species-specific multiplex PCR and short-read genomic sequencing. The banding pattern of the multiplex PCR indicated that the worm was T. murrelli, and the sequence of the mitochondrial Cytochrome c oxidase 1 gene and the ribosomal 18S ribosomal RNA, Internal Transcribed Spacer 1, 5.8S ribosomal RNA, Internal Transcribed Spacer 2, and 28S ribosomal RNA confirmed the diagnosis. This is the first report of T. murrelli in gray foxes that includes assays for assigning parasite species. This report confirms suspected data from surveys conducted over 30 yr ago and establishes a new host record for T. murrelli.

Potential Trimethylamine (TMA)-Producing Bacteria in patients with chronic kidney disease undergoing hemodialysis.

Trimethylamine (TMA), produced by gut microbiota, is the precursor of trimethylamine-N-oxide (TMAO), a uremic toxin that accumulates in patients with chronic kidney disease (CKD). Elevated TMAO plasma levels are associated with cardiovascular complications and CKD progression. To evaluate the association between gut microbiota composition and TMAO plasma levels in CKD patients undergoing hemodialysis (HD). This is a cross-sectional study with 25 patients evaluated (60% female, 53 (18) years, body mass index (BMI) 25.8 (6.75)Kg/m2). They were divided into two groups according to their TMAO plasma levels: normal (≤ 7.4μM) and high (> 7.4μM). Uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS), and indol acetic acid (IAA) were measured with RP-HPLC, and TMAO plasma levels were quantified using LC-MS/MS. Fecal DNA was extracted with a commercial kit, PCR amplified the V4 region of the 16S rRNA gene, and short-read sequencing was performed on the Illumina platform. Dietary intake, anthropometric measurements, and inflammation markers were also evaluated. Nrf2, NF-κB, IL-1β, and NLRP3 mRNA expressions were measured from peripheral blood mononuclear cells (PBMC) using quantitative real-time polymerase chain reaction (qPCR). There were significant positive correlations between TMAO and plasma levels of pCS, NLPR3 inflammasome mRNA expression, serum phosphorus levels, and negative correlations with dietary lipid intake. The group with TMAO > 7.4μM showed an increase in the microbiome abundance of Saccharibacteria (genus incertae sedis), Colidextribacter, Dorea, and Staphylococci genera, and a decrease in abundance in the genera Lachnospira, Lactobacilli, and Victivallis. TMAO plasma level was positively correlated with the abundance of bacteria of the genera Colidextribacter and Helicobacter and was negatively correlated with Sphingomanos, Lachnospira, Streptomyces, and Bacillus genera. Saccharibacteria (genus incertae sedis), Colidextribacter, Dorea, and Staphylococci genera showed higher abundance in patients with high TMAO levels. In addition, we observed that elevated plasma TMAO levels are associated with inflammation markers, dietary lipid intake, and serum phosphorus levels in patients undergoing HD.

Implementing Massive Parallel Sequencing into Biliary Samples Obtained through Endoscopic Retrograde Cholangiopancreatography for Diagnosing Malignant Bile Duct Strictures.

Despite advancements in radiologic, laboratory, and pathological evaluations, differentiating between benign and malignant bile duct strictures remains a diagnostic challenge. Recent developments in massive parallel sequencing (MPS) have introduced new opportunities for early cancer detection and management, but these techniques have not yet been rigorously applied to biliary samples. We prospectively evaluated the Oncomine Comprehensive Assay (OCA) and the Oncomine Pan-Cancer Cell-Free Assay (OPCCFA) using biliary brush cytology and bile fluid obtained via endoscopic retrograde cholangiopancreatography from patients with bile duct strictures. The diagnostic performance of MPS testing was assessed and compared to the pathological findings of biliary brush cytology and primary tissue. Mutations in TP53, BRAF, CTNNB1, SMAD4, and K-/N-RAS identified in biliary brush cytology samples were also detected in the corresponding bile fluid samples from patients with extrahepatic cholangiocarcinoma. These mutations were also identified in the bile fluid samples, but with variant allele frequencies lower than those in the corresponding biliary brush cytology samples. In control patients diagnosed with gallstones, neither the biliary brush cytology samples nor the bile fluid samples showed any pathogenic mutations classified as tier 1 or 2. Our study represents a prospective investigation into the role of MPS-based molecular testing in evaluating bile duct strictures. MPS-based molecular testing shows promise in identifying actionable genomic alterations, potentially enabling the stratification of patients for targeted chemotherapeutic treatments. Future research should focus on integrating OCA and OPCCFA testing, as well as similar MPS-based assays, into existing surveillance and management protocols for patients with bile duct strictures.

An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing

Colorectal cancer (CRC) ranks as the second leading cause of cancer deaths globally. In recent years, short-read single-cell RNA sequencing (scRNA-seq) has been instrumental in deciphering tumor heterogeneities. However, these studies only enable gene-level quantification but neglect alterations in transcript structures arising from alternative end processing or splicing. In this study, we integrated short- and long-read scRNA-seq of CRC samples to build an isoform-resolution CRC transcriptomic atlas. We identified 394 dysregulated transcript structures in tumor epithelial cells, including 299 resulting from various combinations of splicing events. Second, we characterized genes and isoforms associated with epithelial lineages and subpopulations exhibiting distinct prognoses. Among 31,935 isoforms with novel junctions, 330 were supported by The Cancer Genome Atlas RNA-seq and mass spectrometry data. Finally, we built an algorithm that integrated novel peptides derived from open reading frames of recurrent tumor-specific transcripts with mass spectrometry data and identified recurring neoepitopes that may aid the development of cancer vaccines.

Genomics resources for the Rapa Nui (Eastern Island) spiny lobster Panulirus pascuensis (Crustacea: Decapoda: Achelata)

BackgroundThe Easter Island spiny lobster Panulirus pascuensis (Reed, 1954) or ‘Ura’ in the Rapa Nui language, is a little known species native to the south eastern Pacific Ocean, distributed along the coasts of Easter Island, Pitcairn Island, and the Salas y Gómez Ridge. In Easter Island, P. pascuensis is the target of a small and profitable and probably overexploited fishery. In this study, we profited from a series of bioinformatic analyses to mine biological insight from low-pass short-read next generation sequencing datasets; we have estimated genome size and ploidy in P. pascuensis using a k-mer strategy, discovered, annotated, and quantified mobile elements in the nuclear genome, assembled the 45S rRNA nuclear DNA cassette and mitochondrial chromosome, and explored the phylogenetic position of P. pascuensis within the genus Panulirus using the signal retrieved from translated mitochondrial protein coding genes.ResultsK-mer analyses predicted P. pascuensis to be diploid with a haploid genome size ranging between 2.75 Gbp (with k-mer = 51) and 3.39 Gbp (with k-mer = 18). In P. pascuensis, repetitive elements comprise at least a half and a maximum of three fourths of the nuclear genome. Almost a third (64.94%) of the repetitive elements present in the studied nuclear genome were not assigned to any known family of transposable elements. Taking into consideration only annotated repetitive elements, the most abundant were classified as Long Interspersed Nuclear Elements (22.81%). Less common repetitive elements included Long Terminal Repeats (2.88%), Satellite DNA (2.66%), and DNA transposons (2.45%), among a few others. The 45S rRNA DNA cassette of P. pascuensis was partially assembled into two contigs. One contig, 2,226 bp long, encoded a partially assembled 5′ ETS the entire ssrDNA (1,861 bp), and a partial ITS1. A second contig, 6,714 bp long, encoded a partially assembled ITS1, the entire 5.8S rDNA (158 bp), the entire ITS2, the entire lsrDNA (4,938 bp), and a partial 3′ ETS (549 bp). The mitochondrial genome of P. pascuensis was 15,613 bp long and contained 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes (12S ribosomal RNA [rrnS] and 16S ribosomal RNA [rrnL]). A phylomitogenomic analysis based on PCGs retrieved Panulirus pascuensis as sister to a fully supported clade comprising P. cygnus and P. longipes.ConclusionWe expect that the information generated in this study will guide the assembly of a chromosome-level nuclear genome for P. pascuensis in the near future. The newly assembled 45S rRNA nuclear DNA cassette and mitochondrial chromosome can support bioprospecting and biomonitoring of P. pascuensis using environmental DNA. The same elements can help to survey the public market place and detect mislabelling of this and other spiny lobsters. Overall, the genomic resources generated in this study will aid in supporting fisheries management and conservation strategies in this iconic spiny lobster that is likely experiencing overexploitation.

Maple syrup urine disease diagnosis in Brazilian patients by massive parallel sequencing

Biallelic pathogenic variants cause maple syrup urine disease (MSUD) in one of the branched-chain α-keto acid dehydrogenase (BCKDH) complex genes (BCKDHA, BCKDHB, DBT, DLD, and PPM1K) leading to the accumulation of leucine, isoleucine, and valine. This study aimed to perform a molecular diagnosis of Brazilian patients with MSUD using gene panels and massive parallel sequencing. Eighteen Brazilian patients with a biochemical diagnosis of MSUD were analyzed by massive parallel sequencing in the Ion PGM Torrent Server using a gene panel with the BCKDHA, BCKDHB, and DBT genes. The American College of Medical Genetics and Genomics guidelines were used to determine variant pathogenicity. Thirteen patients had both variants found by massive parallel sequencing, whereas 3 patients had only one variant found. In 2 patients, the variants were not found by this analysis. These 5 patients required additional Sanger sequencing to confirm their genotype. Twenty-five pathogenic variants were identified in the 3 MSUD-related genes (BCKDHA, BCKDHB, and DBT). Most variants were present in the BCKDHB gene, and no common variants were found. Nine novel variants were observed: c.922 A > G, c.964C > A, and c.1237 T > C in the BCKDHA gene; and c.80_90dup, c.384delA, c.478 A > T, c.528C > G, c.977 T > C, and c.1039-2 A > G in the BCKDHB gene. All novel variants were classified as pathogenic. Molecular modeling of the novel variants indicated that the binding of monomers was affected in the BCKDH complex tetramer, which could lead to a change in the stability and activity of the enzyme. Massive parallel sequencing with targeted gene panels seems to be a cost-effective method that can provide a molecular diagnosis of MSUD.

Next-generation sequencing in Charcot-Marie-Tooth: a proposal for improvement of ACMG guidelines for variant evaluation

BackgroundThe application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The...

Contributions of Long-Read Sequencing for the Detection of Antimicrobial Resistance.

In the context of increasing antimicrobial resistance (AMR), whole-genome sequencing (WGS) of bacteria is considered a highly accurate and comprehensive surveillance method for detecting and tracking the spread of resistant pathogens. Two primary sequencing technologies exist: short-read sequencing (50-300 base pairs) and long-read sequencing (thousands of base pairs). The former, based on Illumina sequencing platforms (ISPs), provides extensive coverage and high accuracy for detecting single nucleotide polymorphisms (SNPs) and small insertions/deletions, but is limited by its read length. The latter, based on platforms such as Oxford Nanopore Technologies (ONT), enables the assembly of genomes, particularly those with repetitive regions and structural variants, although its accuracy has historically been lower. We performed a head-to-head comparison of these techniques to sequence the K. pneumoniae VS17 isolate, focusing on blaNDM resistance gene alleles in the context of a surveillance program. Discrepancies between the ISP (blaNDM-4 allele identified) and ONT (blaNDM-1 and blaNDM-5 alleles identified) were observed. Conjugation assays and Sanger sequencing, used as the gold standard, confirmed the validity of ONT results. This study demonstrates the importance of long-read or hybrid assemblies for accurate carbapenemase resistance gene identification and highlights the limitations of short reads in the context of gene duplications or multiple alleles. In this proof-of-concept study, we conclude that recent long-read sequencing technology may outperform standard short-read sequencing for the accurate identification of carbapenemase alleles. Such information is crucial given the rising prevalence of strains producing multiple carbapenemases, especially as WGS is increasingly used for epidemiological surveillance and infection control.

Deciphering glial contributions to CSF1R-related disorder via single-nuclear transcriptomic profiling: a case study

CSF1R-related disorder (CSF1R-RD) is a neurodegenerative condition that predominantly affects white matter due to genetic alterations in the CSF1R gene, which is expressed by microglia. We studied an elderly man with a hereditary, progressive dementing disorder of unclear etiology. Standard genetic testing for leukodystrophy and other neurodegenerative conditions was negative. Brain autopsy revealed classic features of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), including confluent white matter degeneration with axonal spheroids and pigmented glial cells in the affected white matter, consistent with CSF1R-RD. Subsequent long-read sequencing identified a novel deletion in CSF1R that was not detectable with short-read exome sequencing. To gain insight into potential mechanisms underlying white matter degeneration in CSF1R-RD, we studied multiple brain regions exhibiting varying degrees of white matter pathology. We found decreased CSF1R transcript and protein across brain regions, including intact white matter. Single nuclear RNA sequencing (snRNAseq) identified two disease-associated microglial cell states: lipid-laden microglia (expressing GPNMB, ATG7, LGALS1, LGALS3) and inflammatory microglia (expressing IL2RA, ATP2C1, FCGBP, VSIR, SESN3), along with a small population of CD44+ peripheral monocyte-derived macrophages exhibiting migratory and phagocytic signatures. GPNMB+ lipid-laden microglia with ameboid morphology represented the end-stage disease microglia state. Disease-associated oligodendrocytes exhibited cell stress signatures and dysregulated apoptosis-related genes. Disease-associated oligodendrocyte precursor cells (OPCs) displayed a failure in their differentiation into mature myelin-forming oligodendrocytes, as evidenced by upregulated LRP1, PDGFRA, SOX5, NFIA, and downregulated NKX2-2, NKX6.2, SOX4, SOX8, TCF7L2, YY1, ZNF488. Overall, our findings highlight microglia–oligodendroglia crosstalk in demyelination, with CSF1R dysfunction promoting phagocytic and inflammatory microglia states, an arrest in OPC differentiation, and oligodendrocyte depletion.