Generation Sequencing Technologies Research Articles

Characterization of mitochondrial DNA mutations in colorectal cancer progression by in silico approach and use as potential biomarkers for diagnosis and prognosis

BackgroundMitochondrial DNA variants are significant contributors to cancer progression, as evidenced by numerous findings. This study focuses on characterizing mitochondrial DNA mutations in colorectal cancer progression and their potential as biomarkers.MethodologyNext generation sequencing technology was employed to analyze mitochondrial DNA variants in tumor and adjacent normal tissues from 25 patients with colon/rectal cancer. In silico prediction tools (SIFT, Polyphen2, Mutation Assessor, and SNP&GO) were utilized to assess the pathogenicity of these variants. Additionally, homology modeling of mutated protein structures was conducted, and molecular dynamic simulations were performed to assess the impact of mutation on protein function.ResultsEighteen variants were identified across most tumor tissue samples, located in genes from Complex I, IV, and V. Among the identified variants, the V302M and S461 mutations in the MT-ND5 gene and L137F and L220P mutations in the ATP6 gene were predicted to be deleterious, potentially affecting protein function. 3D structural analysis of both wild-type and mutant proteins of MT-ND5 revealed changes in flexibility for the V302M and S461G mutations. The MT-ATP6 mutations L135F and L220P disrupt the interactions with surrounding residues and affect the overall function of protein. Further changes in protein dynamics of the mutated proteins by molecular dynamic simulations also indicate the effects; the mutations have on protein function.ConclusionMT-ND5 and MT-ATP6 variants could serve as potential biomarkers and drug targets in colorectal cancer. This study underscores the significance of mitochondrial DNA variants in cancer progression.

Identification of Genetic Variants Associated with Hereditary Thoracic Aortic Diseases (HTADs) Using Next Generation Sequencing (NGS) Technology and Genotype-Phenotype Correlations.

Hereditary thoracic aorta diseases (HTADs) are a heterogeneous group of rare disorders whose major manifestation is represented by aneurysm and/or dissection frequently located at the level of the ascending thoracic aorta. The diseases have an insidious evolution and can be encountered as an isolated manifestation or can also be associated with systemic, extra-aortic manifestations (syndromic HTADs). Along with the development of molecular testing technologies, important progress has been made in deciphering the heterogeneous etiology of HTADs. The aim of this study is to identify the genetic variants associated with a group of patients who presented clinical signs suggestive of a syndromic form of HTAD. Genetic testing based on next-generation sequencing (NGS) technology was performed using a gene panel (Illumina TruSight Cardio Sequencing Panel) or whole exome sequencing (WES). In the majority of cases (8/10), de novo mutations in the FBN1 gene were detected and correlated with the Marfan syndrome phenotype. In another case, a known mutation in the TGFBR2 gene associated with Loeys-Dietz syndrome was detected. Two other pathogenic heterozygous variants (one de novo and the other a known mutation) in the SLC2A10 gene (compound heterozygous genotype) were identified in a patient diagnosed with arterial tortuosity syndrome (ATORS). We presented the genotype-phenotype correlations, especially related to the clinical evolution, highlighting the particularities of each patient in a family context. We also emphasized the importance of genetic testing and patient monitoring to avoid acute aortic events.

Developmental and validation of a novel small and high-efficient panel of microhaplotypes for forensic genetics by the next generation sequencing

BackgroundIn the domain of forensic science, the application of kinship identification and mixture deconvolution techniques are of critical importance, providing robust scientific evidence for the resolution of complex cases. Microhaplotypes, as the emerging class of genetic markers, have been widely studied in forensics due to their high polymorphisms and excellent stability.Results and discussionIn this research, a novel and high-efficient panel integrating 33 microhaplotype loci along with a sex-determining locus was developed by the next generation sequencing technology. In addition, we also assessed its forensic utility and delved into its capacity for kinship analysis and mixture deconvolution. The average effective number of alleles (Ae) of the 33 microhaplotype loci in the Guizhou Han population was 6.06, and the Ae values of 30 loci were greater than 5. The cumulative power of discrimination and cumulative power of exclusion values of the novel panel in the Guizhou Han population were 1-5.6 × 10− 43 and 1-1.6 × 10− 15, respectively. In the simulated kinship analysis, the panel could effectively distinguish between parent-child, full-sibling, half-sibling, grandfather-grandson, aunt-nephew and unrelated individuals, but uncertainty rates clearly increased when distinguishing between first cousins and unrelated individuals. For the mixtures, the novel panel had demonstrated excellent performance in estimating the number of contributors of mixtures with 1 to 5 contributors in combination with the machine learning methods.ConclusionsIn summary, we have developed a small and high-efficient panel for forensic genetics, which could provide novel insights into forensic complex kinships testing and mixture deconvolution.

CannSeek? Yes we Can! Anopen-source single nucleotide polymorphism database and analysis portal for Cannabis sativa.

The CannSeek portal is available at https://icgrc.info/cannseek, https://icgrc.info/genotype_viewer.

Characterization of complete mitochondrial genome of Periclimenes brevicarpalis (Decapoda: Palaemonidae)

Periclimenes brevicarpalis is widely distributed in the Indo West Pacific oceans. The mitochondrial genome of P. brevicarpalis was sequenced and assembled firstly by next generation sequencing technology in our study. The complete mitochondrial genome of P. brevicarpalis was 16,673 bp in size, consisted of 22 transfer RNA genes, 13 protein coding genes and two ribosomal RNA genes. The contents of the four bases were C (25.62%), T (28.73%), A (31.64%), and G (14.01%). The result of phylogenetic analysis showed that P. brevicarpaliswas clustered with Anchistus australis. In conclusion, our research provided valuable data for phylogenetic analysis of the Palaemonidae family as the first report about mitochondrial genomes in Periclimenes.

Comparative Analyses of Lycodon rufozonatus and Lycodon rosozonatus Gut Microbiota in Different Regions.

The interactions between hosts and the gut microbiota are intricate and can significantly affect the ecology and evolution of both parties. Various host traits, including taxonomy, diet, social behaviour, and external factors such as prey availability and the local environment, all play an important role in shaping composition and diversity of the gut microbiogta. In this study, we explored the impact of intestinal microorganisms on the host in adapting to their respective ecological niches in two species of snakes. We collected feces from Lycodon rufozonatus and Lycodon rosozonatus from different geographical locations and used 16S rRNA gene sequencing technology to sequence the v3-v4 region. The results revealed that there was no significant difference in the alpha diversity of intestinal microorganisms between L. rufozonatus and L. rosozonatus. The gut microbiota of all individuals comprised four main phyla: Pseudomonadota, Bacteroidota, Bacillota, and Actinomycetota. At the genus level, the genus Salmonella dominated the enterobacterial microbiota in the samples from Hainan, while there was no obvious dominant genus in the enterobacterial microbiota of the samples from the other four localities. Comparative analysis of enzyme families annotated to the gut microbiota between L. rufozonatus and L. rosozonatus from the four sampling regions by CAZy carbohydrate annotation revealed that nine enzyme families differed significantly in terms of glycoside hydrolases (GHs). In addition, we compared the composition of gut microbial communities between L. rufozonatus and L. rosozonatus and investigated the impact of the differences on their functions. Our results will provide insights into the coevolution of host and gut microbes.

Resistance Induction in Two Alfalfa Varieties to Bean Leafroll Virus and Citrus Excortis Viroid Using Some Botanicals and Amino Acids Preparations

Many researches have been conducted so far in the topic of controlling the Bean leafroll virus and Citrus exocortis viroid. This study was conducted in a private farm at Balad district from September 1, 2023, to January 6, 2024, and aimed to evaluate the efficacy of azolla and clover plant extract, fosetyl aluminum pesticide, and Debal against Bean leafroll virus in the presence of Citrus exocortis viroid in diseased alfalfa. The diagnosis was confirmed for the whole genome of the virus and viroid under study using next generation sequencing technology. The resulting sequences were compared in the International Gene Bank database NCBI. The virus was the 1st record registered in Iraq, which was registered with a accession number PP889325, and the viroid genome was registered for the first time in the NCBI database with an accession number PP869624. Findings of the field experiment showed that the treatments were effective in reducing infection rates and disease severity beside increasing plant defense enzymes (catalase, polyphenol oxidase, and peroxidase) in virus infected alfalfa. Debal treatment, individual or in combination, recorded the highest rate leaf content of catalase (8.00), polyphenol oxidase (3.70), and peroxidase (4.13), with values significantly differed from infected untreated control treatment that recorded 3.33, 0.91, and 0.81, respectively.

WIMOAD: Weighted Integration of Multi-Omics data for Alzheimer's Disease (AD) Diagnosis.

As the most common subtype of dementia, Alzheimer's disease (AD) is characterized by a progressive decline in cognitive functions, especially in memory, thinking, and reasoning ability. Early diagnosis and interventions enable the implementation of measures to reduce or slow further regression of the disease, preventing individuals from severe brain function decline. The current framework of AD diagnosis depends on A/T/(N) biomarkers detection from cerebrospinal fluid or brain imaging data, which is invasive and expensive during the data acquisition process. Moreover, the pathophysiological changes of AD accumulate in amino acids, metabolism, neuroinflammation, etc., resulting in heterogeneity in newly registered patients. Recently, next generation sequencing (NGS) technologies have found to be a non-invasive, efficient and less-costly alternative on AD screening. However, most of existing studies rely on single omics only. To address these concerns, we introduce WIMOAD, a weighted integration of multi-omics data for AD diagnosis. WIMOAD synergistically leverages specialized classifiers for patients' paired gene expression and methylation data for multi-stage classification. The resulting scores were then stacked with MLP-based meta-models for performance improvement. The prediction results of two distinct meta-models were integrated with optimized weights for the final decision-making of the model, providing higher performance than using single omics only. Remarkably, WIMOAD achieves significantly higher performance than using single omics alone in the classification tasks. The model's overall performance also outperformed most existing approaches, highlighting its ability to effectively discern intricate patterns in multi-omics data and their correlations with clinical diagnosis results. In addition, WIMOAD also stands out as a biologically interpretable model by leveraging the SHapley Additive exPlanations (SHAP) to elucidate the contributions of each gene from each omics to the model output. We believe WIMOAD is a very promising tool for accurate AD diagnosis and effective biomarker discovery across different progression stages, which eventually will have consequential impacts on early treatment intervention and personalized therapy design on AD.

Assessment of lower respiratory tract microbiota associated with pulmonary tuberculosis in children.

The respiratory microbiota plays a crucial role in the development of tuberculosis (TB). While existing research has underscored imbalances in the respiratory microbiota of adult patients with TB, information regarding the lower respiratory tract (LRT) microbiota in pediatric patients with TB remains scarce. We employed 16S rRNA gene sequencing technology to investigate the LRT microbial communities of 85 children of different ages with active TB of different severities, 33 children with infectious diseases other than TB, and 48 sex- and age-matched healthy children. A marked imbalance in the respiratory microbiota was observed in children with TB, highlighted by reduced alpha diversity and a distinct microbial community structure. Comparative analysis indicated that patients with severe TB exhibited lower Neisseria levels than those with non-severe TB (1.01% vs. 3.93%, respectively; p = .02). Streptococcus and Gemella levels were lower in bacteriologically confirmed TB cases compared with clinically diagnosed cases, and higher in healthy children younger than 10 years old than in the older group. Spearman correlation analysis demonstrated significant associations between the microbiota of the LRT and cytokine concentrations in the sputum of children with TB (e.g., an inverse correlation between Veillonella and interleukin-17A). TB induced significant dysbiosis in the LRT microbiota of children that was associated with disease severity and the immunological response in the respiratory tract. Our findings may offer a deeper understanding of the role of the respiratory microbiome in TB pathogenesis and progression.

Current Treatment Methods for Charcot-Marie-Tooth Diseases.

Charcot-Marie-Tooth (CMT) disease, the most common inherited neuromuscular disorder, exhibits a wide phenotypic range, genetic heterogeneity, and a variable disease course. The diverse molecular genetic mechanisms of CMT were discovered over the past three decades with the development of molecular biology and gene sequencing technologies. These methods have brought new options for CMT reclassification and led to an exciting era of treatment target discovery for this incurable disease. Currently, there are no approved disease management methods that can fully cure patients with CMT, and rehabilitation, orthotics, and surgery are the only available treatments to ameliorate symptoms. Considerable research attention has been given to disease-modifying therapies, including gene silencing, gene addition, and gene editing, but most treatments that reach clinical trials are drug treatments, while currently, only gene therapies for CMT2S have reached the clinical trial stage. In this review, we highlight the pathogenic mechanisms and therapeutic investigations of different subtypes of CMT, and promising therapeutic approaches are also discussed.

In silico analysis of the effects of non-synonymous SNPs associated with human GSK3B gene on its structure and function

Single Nucleotide Polymorphisms (SNPs) are abundantly identified by next generation sequencing (NGS) technology. Glycogen synthase kinase-3 beta (GSK3B), a widely expressed protein kinase, plays pivotal roles in cellular pathways. However, study on SNPs associated with GSK3B and their functional consequences is lacking. In this study, we analysed non-synonymous SNPs of GSK3B gene and their implications using computational tools. From NCBI dbSNP, 103,087 SNPs of GSK3B were initially gathered, later narrowed down to 255 unique nsSNPs. Around one-third of the nsSNPs resulted in charge and polarity change of the amino acids of the protein. 41 nsSNPs were found to significantly alter the stability of GSK3B protein (ΔΔG ≤ -1 or ≥ 1 kcal/mol) and few of them also affected the disorderness at the mutation site. Evolutionary conservation of the nsSNPs in the protein revealed 25 nsSNP may be deleterious to GSK3B protein function. Finally, 4 critical nsSNPs (Y161C, R167G, P225L and Y234D) were identified that can significantly alter both the stability and function of GSK3B. Furthermore, this study predicted 60 post-translational modification sites in GSK3B among which 26 sites contained nsSNPs. Interestingly, 7 upstream ORFs (uORFs) with high ribosomal occupancy were also detected in GSK3B mRNA that can reduce the expression of GSK3B protein. Altogether, this study has employed various in silico methods to characterize GSK3B nsSNPs, but they have limitations. These tools often overlook the cellular context, interacting partners, PTMs and the dynamic nature of proteins, which can affect protein behaviour and function. Despite these limitations, in silico tools are valuable for initial screening and prioritizing SNPs. The prioritized SNPs obtained in this study (Y161C, R167G, P225L and Y234D) should be experimentally validated using techniques like genome editing, biochemical assays, interactome analysis in cell lines and animal models to confirm their biological relevance.Clinical trial registration: Not Applicable.

A 10-Year Review on Advancements in Identifying and Treating Intellectual Disability Caused by Genetic Variations.

Intellectual disability (ID) is a prevalent neurodevelopmental disorder characterized by neurodevelopmental defects such as the congenital impairment of intellectual function and restricted adaptive behavior. However, genetic studies have been significantly hindered by the extreme clinical and genetic heterogeneity of the subjects under investigation. With the development of gene sequencing technologies, more genetic variations have been discovered, assisting efforts in ID identification and treatment. In this review, the physiological basis of gene variations in ID is systematically explained, the diagnosis and therapy of ID is comprehensively described, and the potential of genetic therapies and exercise therapy in the rehabilitation of individuals with intellectual disabilities are highlighted, offering new perspectives for treatment approaches.

Effects of fecal microbiota transplantation combined with selenium on intestinal microbiota in mice with colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the world. With the development of high-throughput gene sequencing technology, homeostasis imbalance of the intestinal microbiota has been proven to play a key role in the pathogenesis of CRC. Furthermore, fecal bacteria transplantation (FMT) has been shown to alter the intestinal microecology, and is potentially an effective treatment for CRC. Sodium selenite plays an important role in anticancer adjuvant therapy due to its high pro-oxidation characteristics. In this study, a murine CRC tumor model was induced by AOM/DSS, and CRC mice were treated by FMT, sodium selenite, and FMT combined with sodium selenite. The results showed that FMT, sodium selenite, and FMT combined with sodium selenite inhibited the occurrence of CRC in mice, increased the abundance of beneficial intestinal bacteria, produced different microorganisms, and changed the metabolic pathways of the intestinal microbiota. In summary, FMT, sodium selenite, and FMT combined with sodium selenite can inhibit the occurrence of CRC by increasing the abundance of beneficial bacteria and regulating phenotypes and metabolic pathways. Notably, the effect of FMT combined with sodium selenite in reducing the number of tumors, protecting intestinal tissues, and restoring the diversity and richness of the intestinal microbiota is superior to that of FMT alone or sodium selenite alone. The results of this study provide new ideas for the application of FMT and selenium in the treatment of CRC.

Viral Co-Infection Diagnosis in Cervical Samples Using Next Generation Sequencing Technology

Viral Co-Infection Diagnosis in Cervical Samples Using Next Generation Sequencing Technology

Resistance to ACCase and ALS-inhibiting herbicides detected in targeted sampling of Lolium rigidum (rigid ryegrass) populations from cereal crops in Morocco, Algeria, and Tunisia

Lolium rigidum Gaud. (rigid ryegrass) is one of the most widespread weeds in cereal crops in Morocco, Algeria and Tunisia. This weed has evolved resistance to various herbicide modes of action in this region. ACCase and ALS inhibiting herbicides are mainly used in the major-cereal growing regions to control rigid ryegrass. Through a questionnaire, regions where farmers reported less control of herbicide treatments were registered in the three Maghreb countries. Registered fields were visited for collection and 75 field populations were screened with two ACCase and two ALS herbicides. Target site resistance (TSR) was diagnosed using Illumina Next Generation Sequencing (NGS) technology. The sensitivity bioassay results revealed over 60% of sampled populations to be resistant to pinoxaden and/or clodinafop and about 40% to be resistant to iodosulfuron + mesosulfuron and/or pyroxsulam. In addition, 53% of populations displayed resistance (R) to the two herbicide modes of action tested among the regions. In total, 16 ACCase and 11 ALS mutant alleles were identified, carrying out an amino-acid substitution and conferring herbicide resistance in 3700 of the analyzed plants. Most ACCase and ALS mutations were detected at codons Ile1781 and Pro197, respectively. Not only does this study demonstrate the presence of both cross and multiple resistance, it also highlights the non-ACCase and non-ALS -based resistance mechanisms that could confer resistance to herbicides with different modes of action which complicates the resistance management strategies. In the three Maghreb countries, this challenge is even more prominent due to few modes of action being available for rigid ryegrass control due to low-cost market and the prevalence of generic herbicides.

Effects of semaglutide on gut microbiota, cognitive function and inflammation in obese mice.

This study aims to investigate the effects of semaglutide on gut microbiota, cognitive function, and inflammation in obese mice. Twenty-four C57BL/6J male mice were randomly assigned to three groups: a normal-chow diet group (NCD, n=8), high-fat diet group (HFD, n=8), and HFD+semaglutide group (Sema, n=8). The mice were fed a HFD to establish an animal model of obesity and then administered with semaglutide or saline for 12 weeks. Cognitive function was assessed using the Morris water maze test. Serum pro-inflammatory cytokines were measured. 16S rRNA gene sequencing technology was used to explore gut microbiota characteristics in obese mice. Obese mice showed significant cognitive impairment and inflammation. Semaglutide improved cognitive function and attenuated inflammation induced by a HFD diet. The abundance of gut microbiota was significantly changed in the HFD group, including decreased Akkermansia, Muribaculaceae, Coriobacteriaceae_UCG_002, Clostridia_UCG_014 and increased Romboutsia, Dubosiella, Enterorhabdus. Whereas semaglutide could dramatically reverse the relative abundance of these gut microbiota. Correlation analysis suggested that cognitive function was positively correlated with Muribaculaceae and Clostridia_UCG_014, and negatively associated with Romboutsia and Dubosiella. Romboutsia was positively correlated with TNFα, IL-6 and IL-1β. While Clostridia_UCG_014 was negatively related to TNFα, IL-6 and IL-1β. For the first time semaglutide displayed different regulatory effects on HFD-induced gut microbiota dysbiosis. Semaglutide could regulate the structure and composition of gut microbiota associated with cognitive function and inflammation. Thus, affecting gut microbiota might be a potential mechanism of semaglutide in attenuating cognitive function and inflammation.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

Cerebral venous sinus thrombosis and SCN1A, a novel association?

Cerebral venous sinus thrombosis (CVST) is a rare cause of stroke. Acquired and inherited prothrombotic conditions are the most common risk factors for CVST. Sometimes, an etiology is not found. Wide utilization of next generation sequencing technologies in clinical practice may lead to identification of risk factors other than those classically associated with CVST. This retrospective clinical-laboratory observational study has a reference patient who presented with CVST as an adolescent. Work up for prothrombotic conditions showed high homocysteine level secondary to homozygosity for a common polymorphism, c.677C > T in the methylenetetrahydrofolate reductase (MTHFR) gene. His older unaffected brother has a similar MTHFR genotype and high homocysteine. The whole exome sequencing revealed a likely pathogenic variant in the sodium voltage gated channel, alpha subunit 1(SCN1A) gene. CVST is a multifactorial disease. Prothrombotic conditions are the most common risk factors for CVST. High homocysteine due to the common MTHFR polymorphisms was previously attributed to various thrombotic conditions including CVST. Although high homocysteine due to MTHFR polymorphism may be a contributing factor, additional risk factors such as blood flow abnormalities during SCN1A related seizures may be needed for thrombosis.

Progress of the Detection Methods for SARS-CoV-2.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a respiratory illness, characterized by symptoms such as fever, dry cough, and drowsiness. This virus is highly contagious, has significant mutation rates, and induces infection despite vaccination. Its widespread prevalence has profoundly impacted global economies, societies, and daily life. In response to these challenges, researchers have committed themselves to advancing rapid and cost-effective diagnostic technologies, holding substantial importance for the rapid evolution of global diagnostic capabilities. Nonetheless, various detection methods diverge in principles, sensitivity, specificity, and other aspects. Additionally, COVID-19 is not an isolated event, but part of a broader history of pandemics in human society. Therefore, this article briefly reviews the existing detection methods of SARS-CoV-2, providing valuable technical insights to diagnose not only SARS-CoV-2 but also other viruses. A search was conducted on PubMed by utilizing keywords such as "SARS-CoV-2 detection", "RT-qPCR detection for SARS-CoV-2", "LFA detection for SARS-CoV-2", "Biosensors detection for SARS-CoV-2", and similar terms. The objective was to compile and summarize relevant articles on these topics. Currently, the real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) stands as a widely employed method for detecting SARS-CoV-2, enabling an accurate detection of viral RNA. Furthermore, the lateral flow assay (LFA) assists in detecting viral antigens and antibodies. Gene sequencing technology primarily facilitates the real-time monitoring of mutated SARS-CoV-2 strains, while biosensors could offer a rapid, economical, sensitive, and precise detection of SARS-CoV-2. These methods provide a strong technical support for the early detection and diagnosis of SARS-CoV-2. This paper offers a concise overview of pathogen detection methods, as molecular biology, and immunological detection techniques, alongside emerging biosensor platforms relevant to SARS-CoV-2, and delineates the strengths and weaknesses of each method.

GuidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel

Since the introduction of next generation sequencing technologies, the field of epigenomics has evolved rapidly. However, most commonly used assays are enrichment-based methods and thus only semi-quantitative. Nucleosome occupancy and methylome sequencing (NOMe-seq) allows for quantitative inference of chromatin states with single locus resolution, but this requires high sequencing depth and is therefore prohibitively expensive to routinely apply to organisms with large genomes. To overcome this limitation, we introduce guidedNOMe-seq, where we combine NOMe profiling with large scale sgRNA synthesis and Cas9-mediated region-of-interest (ROI) liberation. To facilitate quantitative comparisons between multiple samples, we additionally develop an R package to standardize differential analysis of any type of NOMe-seq data. We extensively benchmark guidedNOMe-seq in a proof-of-concept study, dissecting the interplay of ChAHP and CTCF on chromatin. In summary we present a cost-effective, scalable, and customizable target enrichment extension to the existing NOMe-seq protocol allowing genome-scale quantification of nucleosome occupancy and transcription factor binding at single allele resolution.