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Nucleic Acids Research Jun 2024Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma,...
Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.
Topics: DNA Methylation; Humans; Cell-Free Nucleic Acids; CpG Islands; DNA, Single-Stranded; 5-Methylcytosine; Lung Neoplasms; Sulfites; Promoter Regions, Genetic; Sequence Analysis, DNA; Whole Genome Sequencing
PubMed: 38797520
DOI: 10.1093/nar/gkae276 -
Electrophoresis May 2024In forensic science, the demand for precision, consistency, and cost-effectiveness has driven the exploration of next-generation sequencing technologies. This study...
In forensic science, the demand for precision, consistency, and cost-effectiveness has driven the exploration of next-generation sequencing technologies. This study investigates the potential of Oxford Nanopore Sequencing (ONT) Technology for analyzing the HIrisPlex-S panel, a set of 41 single nucleotide polymorphism (SNP) markers used to predict eye, hair, and skin color. Using ONT sequencing, we assessed the accuracy and reliability of ONT-generated data by comparing it with conventional capillary electrophoresis (CE) in 18 samples. The Guppy v6.1 was used as a basecaller, and sample profiles were obtained using Burrows-Wheeler Aligner, Samtools, BCFtools, and Python. Comparing accuracy with CE, we found that 62% of SNPs in ONT-unligated samples were correctly genotyped, with 36% showing allele dropout, and 2% being incorrectly genotyped. In the ONT-ligated samples, 85% of SNPs were correctly genotyped, with 10% showing allele dropout, and 5% being incorrectly genotyped. Our findings indicate that ONT, particularly when combined with ligation, enhances genotyping accuracy and coverage, thereby reducing allele dropouts. However, challenges associated with the technology's error rates and the impact on genotyping accuracy are recognized. Phenotype predictions based on ONT data demonstrate varying degrees of success, with the technology showing high accuracy in several cases. Although ONT technology holds promise in forensic genetics, further optimization and quality control measures are essential to harness its full potential. This study contributes to the ongoing efforts to refine sequence read tuning and improve correction tools in the context of ONT technology's application in forensic genetics.
PubMed: 38794987
DOI: 10.1002/elps.202300252 -
Viruses May 2024Mouse adenoviruses (MAdV) play important roles in studying host-adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An...
Mouse adenoviruses (MAdV) play important roles in studying host-adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.
Topics: Animals; Mice; Genetic Vectors; Virus Replication; Plasmids; Adenoviridae; NIH 3T3 Cells; Cloning, Molecular; Genes, Reporter
PubMed: 38793642
DOI: 10.3390/v16050761 -
Biosensors May 2024Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease...
Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL, 0.77 ng∙μL, and 1.1 ng∙μL, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.
Topics: Fluorescence Resonance Energy Transfer; Aptamers, Nucleotide; Humans; Exosomes; Biosensing Techniques; Biomarkers; Nucleic Acid Amplification Techniques; Tetraspanin 30
PubMed: 38785707
DOI: 10.3390/bios14050233 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
PLoS Genetics May 2024How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter...
How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.
Topics: Enhancer Elements, Genetic; Humans; Promoter Regions, Genetic; Chromatin; Estrogens; Transcription, Genetic; MCF-7 Cells; Breast Neoplasms; Female; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; In Situ Hybridization, Fluorescence; Receptors, Estrogen; Transcriptional Activation; Estrogen Receptor alpha
PubMed: 38781242
DOI: 10.1371/journal.pgen.1011277 -
Frontiers in Cell and Developmental... 2024Mammalian genomic DNA is packed in a small nucleus, and its folding and organization in the nucleus are critical for gene regulation and cell fate determination. In... (Review)
Review
Mammalian genomic DNA is packed in a small nucleus, and its folding and organization in the nucleus are critical for gene regulation and cell fate determination. In interphase, chromosomes are compartmentalized into certain nuclear spaces and territories that are considered incompatible with each other. The regulation of gene expression is influenced by the epigenetic characteristics of topologically associated domains and A/B compartments within chromosomes (intrachromosomal). Previously, interactions among chromosomes detected via chromosome conformation capture-based methods were considered noise or artificial errors. However, recent studies based on newly developed ligation-independent methods have shown that inter-chromosomal interactions play important roles in gene regulation. This review summarizes the recent understanding of spatial genomic organization in mammalian interphase nuclei and discusses the potential mechanisms that determine cell identity. In addition, this review highlights the potential role of inter-chromosomal interactions in early mouse development.
PubMed: 38774644
DOI: 10.3389/fcell.2024.1397807 -
Environmental and Molecular Mutagenesis 2024As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a...
As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.
Topics: Animals; Mice; DNA; Male; Female; Liver; Cerebellum; Mice, Inbred C57BL; DNA Ligases; DNA Repair
PubMed: 38767089
DOI: 10.1002/em.22602 -
BioRxiv : the Preprint Server For... May 2024DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair...
DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3'-oxidative damage incorporated by DNA polymerase (pol) β at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3'-8oxodG and 3'-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3'-8oxodG:A and 3'-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3α (LIG3α) . Finally, we observed that LIG1 and LIG3α seal resulting nick after an incorporation of 8oxorGTP:A by polβ and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, polβ, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.
PubMed: 38766188
DOI: 10.1101/2024.05.06.592774 -
Clinica Chimica Acta; International... Jun 2024Next-generation sequencing (NGS)-based copy number variants (CNVs) have high false-positive rates. The fewer the exons involved, the higher the false-positive rate. A... (Comparative Study)
Comparative Study
BACKGROUND AND AIMS
Next-generation sequencing (NGS)-based copy number variants (CNVs) have high false-positive rates. The fewer the exons involved, the higher the false-positive rate. A CytoScan XON assay was developed to assess exon-level CNVs.
MATERIALS AND METHODS
Twenty-three clinically relevant exon-level CNVs in 20 patient blood samples found in previous NGS studies were compared with the results from the CytoScan XON and multiplex ligation-dependent probe amplification (MLPA).
RESULTS
Fifteen of the 23 exon-level CNVs were consistent with the NGS results. Among these, eight were confirmed using MLPA. In six out of eight discrepancies between the CytoScan Xon and NGS, MLPA was performed, and three were negative, indicating that the CNVs in NGS were false positives. The CytoScan XON exhibits a sensitivity of 72.7% for small exon-level CNVs, along with a specificity of 100%. The assay could not detect the three exon-level CNVs in PKD1 and TSC2 that were detected using both NGS and MLPA. This could be due to the distribution of the probes in some areas, and the CNV-calling regions containing multiple exons.
CONCLUSION
The CytoScan XON assay is a promising complementary tool for the detection of exon-level CNVs, provided that the users carefully examine the distribution of probes and calling regions.
Topics: Humans; DNA Copy Number Variations; High-Throughput Nucleotide Sequencing; Exons
PubMed: 38763467
DOI: 10.1016/j.cca.2024.119703