Long-read Sequencing Research Articles

Unravelling the architecture of major histocompatibility complex class II haplotypes in rhesus macaques.

The regions in the genome that encode components of the immune system are often featured by polymorphism, copy number variation, and segmental duplications. There is a need to thoroughly characterize these complex regions to gain insight into the impact of genomic diversity on health and disease. Here we resolve the organization of complete major histocompatibility complex (MHC) class II regions in rhesus macaques by using a long-read sequencing strategy (Oxford Nanopore Technologies) in concert with adaptive sampling. In particular, the expansion and contraction of the primate DRB-region appear to be a dynamic process that involves the rearrangement of different cassettes of paralogous genes. These chromosomal recombination events are propagated by a conserved pseudogene, DRB6, which features the integration of two retroviral elements. In contrast, the DRA locus appears to be protected from rearrangements, which may be owing to the presence of an adjacently located truncated gene segment, DRB9 With our sequencing strategy, the annotation, evolutionary conservation, and potential function of pseudogenes can be reassessed, an aspect that was neglected by most genome studies in primates. Furthermore, our approach facilitates the characterization and refinement of an animal model essential to study human biology and disease.

CascAGS: Comparative Analysis of SNP Calling Methods for Human Genome Data in the Absence of Gold Standard.

The development of third-generation sequencing has accelerated the boom of single nucleotide polymorphism (SNP) calling methods, but evaluating accuracy remains challenging owing to the absence of the SNP gold standard. The definitions for without-gold-standard and performance metrics and their estimation are urgently needed. Additionally, the possible correlations between different SNP loci should also be further explored. To address these challenges, we first introduced the concept of a gold standard and imperfect gold standard under the consistency framework and gave the corresponding definitions of sensitivity and specificity. A latent class model (LCM) was established to estimate the sensitivity and specificity of callers. Furthermore, we incorporated different dependency structures into LCM to investigate their impact on sensitivity and specificity. The performance of LCM was illustrated by comparing the accuracy of BCFtools, DeepVariant, FreeBayes, and GATK on various datasets. Through estimations across multiple datasets, the results indicate that LCM is well-suitable for evaluating callers without the SNP gold standard, and accurate inclusion of the dependency between variations is crucial for better performance ranking. DeepVariant has a higher sum of sensitivity and specificity than other callers, followed by GATK and BCFtools. FreeBayes has low sensitivity but high specificity. Notably, appropriate sequencing coverage is another important factor for precise callers' evaluation. Most importantly, a web interface for assessing and comparing different callers was developed to simplify the evaluation process.

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

BackgroundMastitis, 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.MethodsOxford-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.ResultsSamples 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.ConclusionsThese 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.

Impact of propionic acid-rich diets on microbial composition of the murine gut microbiome

Propionic acid (PPA), an anti-fungal agent and common food additive, has been shown to induce atypical neurodevelopment in mice, accompanied by gastrointestinal dysfunction potentially resulting from gut dysbiosis. A putative association between dietary PPA exposure and gut dysbiosis is suggested but has not been explored directly. Here, we investigated PPA-associated alteration in gut microbial composition that may result in dysbiosis. Using long-read metagenomic sequencing, gut microbiomes of mice fed an untreated (n=9) or PPA-rich (n=13) diet were sequenced to assess differences in microbial composition and bacterial metabolic pathways. Dietary PPA was associated with an increased abundance of notable taxa, including several species of Bacteroides, Prevotella, and Ruminococcus, whose member species have previously been associated with PPA production. Microbiomes of PPA exposed mice also possessed a greater abundance of pathways related to lipid metabolism and steroid hormone biosynthesis. Our findings demonstrate PPA’s effect in altering the gut microbiota and associated metabolic pathways. These observed changes highlight how preservatives listed as safe for consumption may affect gut microbiome composition with implications for one’s health.

Long-read genome assembly of the insect model organism Tribolium castaneum reveals spread of satellite DNA in gene-rich regions by recurrent burst events.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences that are mostly underrepresented in genome assemblies. Here we combined nanopore long-read sequencing with a reference-guided assembly approach to generate an improved, high-quality genome assembly, TcasONT, of the model beetle Tribolium castaneum Enriched by 45 Mb in repetitive regions, the new assembly comprises almost the entire genome sequence. We use the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs. Unexpectedly, we show the extensive spread of satDNAs in gene-rich regions, including long arrays. The sequence similarity relationships between satDNA monomers and arrays indicate a recent exchange of satDNA arrays between different chromosomes. We propose a scenario of their genome dynamics characterized by repeated bursts of satDNAs spreading through euchromatin, followed by a process of elongation and homogenization of arrays. We find that suppressed recombination on the X Chromosome has no significant effect on the spread of satDNAs but the X rather tolerates the amplification of satDNAs into longer arrays. Analyses of arrays' neighboring regions show a tendency of one satDNA to be associated with transposable-like elements. Using 2D electrophoresis followed by Southern blotting, we prove Cast satDNAs' presence in the fraction of extrachromosomal circular DNA (eccDNA). We point to two mechanisms that enable this satDNA spread to occur: transposition by transposable elements and insertion mediated by eccDNA. The presence of such a large proportion of satDNA in gene-rich regions inevitably gives rise to speculation about their possible influence on gene expression.

A cry for kelp: Evidence for polyphenolic inhibition of Oxford Nanopore sequencing of brown algae.

Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long-read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol-DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel-suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide-ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash-frozen tissue wherever possible for genome sequencing.

The Ribosomal Operon Database: A Full-Length rDNA Operon Database Derived From Genome Assemblies.

Current rDNA reference sequence databases are tailored towards shorter DNA markers, such as parts of the 16/18S marker or the internally transcribed spacer (ITS) region. However, due to advances in long-read DNA sequencing technologies, longer stretches of the rDNA operon are increasingly used in environmental sequencing studies to increase the phylogenetic resolution. There is, therefore, a growing need for longer rDNA reference sequences. Here, we present the ribosomal operon database (ROD), which includes eukaryotic full-length rDNA operons fished from publicly available genome assemblies. Full-length operons were detected in 34.1% of the 34,701 examined eukaryotic genome assemblies from NCBI. In most cases (53.1%), more than one operon variant was detected, which can be due to intragenomic operon copy variability, allelic variation in non-haploid genomes, or technical errors from the sequencing and assembly process. The highest copy number found was 5947 in Zea mays. In total, 453,697 unique operons were detected, with 69,480 operon variant clusters remaining after intragenomic clustering at 99% sequence identity. The operon length varied extensively across eukaryotes, ranging from 4136 to 16,463 bp, which will lead to considerable polymerase chain reaction (PCR) bias during amplification of the entire operon. Clustering the full-length operons revealed that the different parts (i.e., 18S, 28S, and the hypervariable regions V4 and V9 of 18S) provide divergent taxonomic resolution, with 18S, the V4 and V9 regions being the most conserved. The ROD will be updated regularly to provide an increasing number of full-length rDNA operons to the scientific community.

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

GCI: a continuity inspector for complete genome assembly.

Recent advances in long-read sequencing technologies have significantly facilitated the production of high-quality genome assembly. The telomere-to-telomere (T2T) gapless assembly has become the new golden standard of genome assembly efforts. Several recent efforts have claimed to produce T2T-level reference genomes. However, a universal standard is still missing to qualify a genome assembly to be at T2T standard. Traditional genome assembly assessment metrics (N50 and its derivatives) have no capacity in differentiating between nearly T2T assembly and the truly T2T assembly in continuity either globally or locally. Additionally, these metrics are independent of raw reads, making them inflated easily by artificial operations. Therefore, a gaplessness evaluation tool at single-nucleotide resolution to reflect true completeness is urgently needed in the era of complete genomes. Here, we present a tool called Genome Continuity Inspector (GCI), designed to assess genome assembly continuity at single-base resolution, and evaluate how close an assembly is to the T2T level. GCI utilizes multiple aligners to map long reads from various sequencing platforms back to the assembly. By incorporating curated mapping coverage of high-confidence read alignments, GCI identifies potential assembly issues. Meanwhile, it provides GCI scores that quantify overall assembly continuity on the whole genome or chromosome scales. The open-source GCI code is freely available on Github (https://github.com/yeeus/GCI) under the MIT license.

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.

Optical genome mapping of structural variants in Parkinson’s disease-related induced pluripotent stem cells

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants, as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast, larger SVs, which may significantly influence human phenotypes, have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study, we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines, which were not identified by long-read sequencing. Additionally, various exon deletions were confirmed by OGM in the PRKN gene of iPSCs, of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs, no gene fusions, no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary, OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques, which is supportive for OGM’s future use in gene discovery and iPSC line screening.

The alternative splicing landscape of infarcted mouse heart identifies isoform level therapeutic targets

Alternative splicing is an important process that contributes to highly diverse transcripts and protein products, which can affect the development of disease in various organisms. Cardiovascular disease (CVD) represents one of the greatest global threats to humans, particularly acute myocardial infarction (MI) and subsequent ischemic reperfusion (IR) injury, which involve complex transcriptomic changes in heart tissues associated with metabolic reshaping and immunological response. In this study, we used a newly developed ONT full-length transcriptomic approach and performed transcript-resolved differential expression profiling in murine models of MI and IR. We built an analytical pipeline to reliably identify and quantify alternative splicing products (isoforms), expanding on the currently available catalog of isoforms described in mice. The updated alternative splicing landscape included transcripts, genes, and pathways that were differentially regulated during IR and MI. Our study establishes a pipeline to profile highly diverse isoforms using state-of-the-art long-read sequencing, builds a landscape of alternative splicing in the mouse heart during MI and IR.

Investigating the Quantification Capabilities of a Nanopore-Based Sequencing Platform for Food Safety Application via External Standards of Lambda DNA and Lambda Spiked Beef.

Six hundred million cases of disease and roughly 420,000 deaths occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. These methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a relatively new technology that combines DNA-sequencing chemistry and bioinformatics to generate and analyze large amounts of short- or long-read DNA sequences and whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies' MinION sequencer through a shotgun-based sequencing approach. This investigation explored the correlation between known amounts of the analyte (lambda DNA as a pathogenic bacterial surrogate) with data output, in both the presence and absence of a background matrix (Bos taurus DNA). A positive linear correlation was observed between the concentration of analyte and the amount of data produced, number of bases sequenced, and number of reads generated in both the presence and absence of a background matrix. In the presence of bovine DNA, the sequenced data were successfully mapped to the NCBI lambda reference genome. Furthermore, the workflow from pre-extracted DNA to target identification took less than 3 h, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification.

A chromosome-scale genome assembly of the peanut beetle, Ulomoides dermestoides (Coleptera: Tenebrionidae)

Ulomoides dermestoides, a member of the Tenebrionidae family, is known by various common names such as Chinese beetle, Chinese weevil, peanut beetle, cancer beetle, or asthma beetle. Despite its origin in Asia, it has achieved global distribution owing to its perceived medicinal properties. In this study, we employed a combination of Illumina short-read, PacBio long-read, and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to produce a high-quality, chromosome-scale genome assembly of U. dermestoides. The assembled genome spans 253.38 Mb across 11 chromosomes, including the two sex chromosomes, X and Y. Key metrics include contig and scaffold N50 values of 9.83 Mb and 28.62 Mb, respectively, with an estimated genome completeness of 99.62%. Annotation efforts identified 15,553 protein-coding genes and revealed that 48.11% of the genome comprises repetitive sequences. This meticulously assembled genome serves as a pivotal resource for elucidating the genetic determinants underlying the species’ ecological habits and potential medicinal properties.

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair.

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from thirteen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. These data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

SARS-CoV-2 variant survey: Comparison of RT-PCR screening with TGS and variant distribution across two divisions of Bangladesh.

The widespread increase in multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) since 2020 is causing significant health concerns worldwide. While whole-genome sequencing (WGS) has played a leading role in surveillance programs, many local laboratories lack the expertise and resources. Thus, we aimed to investigate the circulating SARS-CoV-2 variants and evaluate the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) for screening and monitoring the emergence of new SARS-CoV-2 variants in Bangladesh. A total of 600 confirmed SARS-CoV-2-positive cases were enrolled either prospectively or retrospectively from two divisions of Bangladesh. The samples were screened by variant RT-PCR targeting five mutations of the spike gene (N501Y, P681R, L452R, E484K, E484Q). A subsample of the study population was also selected for third-generation sequencing (TGS) and the results were compared to the variant RT-PCR screening. An in-depth comparison was made between the two methods in terms of congruence and cost-benefit. Seven variants were detected among samples, with similar distributions of the variants across both divisions. Variant RT-PCR for the targeted mutations lead to a 98.5% call rate; only nine samples failed to be determined. No association was found regarding the demographic features, clinical criteria, or routine RT-PCR Ct values across the variants. The clade diversity of the sequenced subpopulation (n = 99) exhibited similar distributions across the two study sites and other epidemiologic variables. Variant RT-PCR successfully distinguished variants of concern (VOCs) and variants of interest (VOIs); however, 8% discrepancy was observed for the closest lineages. Moreover, the variant RT-PCR represented an ideal balance of cost, time, and accuracy that outweigh their limitations. Based on the strong agreement of variant RT-PCR with TGS, such rapid, easily accessible approaches of rapid strain typing are essential in the context of pandemic responses to guide both treatment decisions and public health measures.

The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

RNA surveillance systems degrade aberrant RNAs that result from defective transcriptional termination, splicing, and polyadenylation. Defective RNAs in the nucleus are recognized by RNA-binding proteins and MTR4, and are degraded by the RNA exosome complex. Here, we detect aberrant RNAs in MTR4-depleted cells using long-read direct RNA sequencing and 3′ sequencing. MTR4 destabilizes intronic polyadenylated transcripts generated by transcriptional read-through over one or more exons, termed 3′ eXtended Transcripts (3XTs). MTR4 also associates with hnRNPK, which recognizes 3XTs with multiple exons. Moreover, the aberrant protein translated from KCTD13 3XT is a target of the hnRNPK-MTR4-RNA exosome pathway and forms aberrant condensates, which we name KCTD13 3eXtended Transcript-derived protein (KeXT) bodies. Our results suggest that RNA surveillance in human cells inhibits the formation of condensates of a defective polyadenylated transcript-derived protein.

A unified framework to analyze transposable element insertion polymorphisms using graph genomes

Transposable elements are ubiquitous mobile DNA sequences generating insertion polymorphisms, contributing to genomic diversity. We present GraffiTE, a flexible pipeline to analyze polymorphic mobile elements insertions. By integrating state-of-the-art structural variant detection algorithms and graph genomes, GraffiTE identifies polymorphic mobile elements from genomic assemblies or long-read sequencing data, and genotypes these variants using short or long read sets. Benchmarking on simulated and real datasets reports high precision and recall rates. GraffiTE is designed to allow non-expert users to perform comprehensive analyses, including in models with limited transposable element knowledge and is compatible with various sequencing technologies. Here, we demonstrate the versatility of GraffiTE by analyzing human, Drosophila melanogaster, maize, and Cannabis sativa pangenome data. These analyses reveal the landscapes of polymorphic mobile elements and their frequency variations across individuals, strains, and cultivars.

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

Tandem repeats (TRs) 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 Pacific Bioscience (PacBio, Sequel II and Revio), otter and TREAT achieve state-of-the-art genotyping and motif characterization accuracy. Applied to clinically relevant TRs, TREAT/otter significantly identify individuals with pathogenic TR expansions. When applied to a case-control setting, we replicate previously reported associations of TRs with Alzheimer's disease, including those near or within APOC1 (P = 2.63 × 10-9), SPI1 (P = 6.5 × 10-3), and ABCA7 (P = 0.04) genes. Finally, we use TREAT/otter to systematically evaluate potential biases when genotyping TRs using diverse ONT and PacBio long-read sequencing data sets. We show that, in rare cases (0.06%), long-read sequencing 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 TRs across different sequencing technologies and with minimal requirements, allowing end-to-end analysis and comparisons of TRs in human genomes, with broad applications in research and clinical fields.

Comprehensive Analysis of Complex Copy Number Variations in the Plasmodium falciparum Genome via Long-Read Sequencing: Implications for Antimalarial Resistance and Broad Genomic Insights

Abstract Introduction/Objective The genome of Plasmodium falciparum, characterized by a high AT content and a propensity for copy number variations (CNVs), presents a model for understanding how genomes adapt to stress. Our research focused on the prevalence and distribution of CNVs highlights long-read sequencing as a tool for detecting complex structural variants in other models. Methods/Case Report P. falciparum parasites were cultured and treated with DSM1 and aphidicolin that induce replication stress. Long-read sequencing was conducted using the Oxford Nanopore Technology Minion, with an emphasis on an optimizing DNA extraction method for preserving intact chromosomes. Visualization of CNVs, including tandem and inverted duplications, was achieved through a custom R Shiny application that allowed both quantitative and qualitative assessments. Results (if a Case Study enter NA) Long read sequencing identified an enrichment of multiple CNV types in treated samples, with a significant enrichment of inverted duplications as well as other types of structural variants. Aphidicolin and DSM1-treated samples demonstrated significant accumulation of these variations compared to controls (2.0 and 5.6-fold, respectively with a p value of < 0.0001 in both treatment groups). These occurred mainly in core genomic regions that do not contain variable gene copies. An increase in the number of inverted duplications may be related to stress response to the treatment and may be a potential adaptive mechanism of the parasite. Our manual single-read visualization technique made it possible to visualize the structure of various CNV types, which provides unique insight on CNVs as they arise in single genomes. Conclusion We hypothesize that inverted duplications represent stalled replication forks, and we are currently assessing their frequency in replicating and non-replicating cells. This study highlights the role of long-read sequencing in detecting complex genomic structures in P. falciparum, which may influence the parasite’s resistance to drug treatment. These findings not only advance our understanding of malaria pathology but also aid our comprehension of how cancer cells might react to drug-induced stress and the mechanisms behind CNV formation in neoplastic cells.