What is the “modified” CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol

Applications in Plant Sciences, Journal Year: 2023, Volume and Issue: 11(3)

Published: May 1, 2023

Cetyltrimethylammonium bromide (CTAB)-based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among species has necessitated optimization. Research articles often cite a "modified" CTAB protocol without explicitly stating how had been altered, creating non-reproducible studies. Furthermore, various modifications that have applied not rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed literature for modified protocols isolation DNA. found every stage modified, we summarized those provide recommendations extraction Future genomic studies will rely on optimized protocols. Our review used, as well here, better standardize extractions, allowing repeatable transparent

Language: Английский

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Balancing read length and sequencing depth: Optimizing Nanopore long‐read sequencing for monocots with an emphasis on the Liliales

Applications in Plant Sciences, Journal Year: 2023, Volume and Issue: 11(3)

Published: May 1, 2023

We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications standard protocols directly impact read length total output. The goal is help those interested in generating data determine which steps may be necessary optimizing output results.

Language: Английский

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Combination of Sample Preservation Approaches and DNA Extraction Methods for Long‐Read Sequencing of Nudibranchs' Genomes

Ecology and Evolution, Journal Year: 2025, Volume and Issue: 15(4)

Published: April 1, 2025

ABSTRACT With the increasing interest in whole genome sequencing of eukaryotes, it is becoming evident that selecting most suitable high molecular weight DNA extraction method crucial for maximizing benefits long‐read technologies. However, many species cannot be processed immediately at sampling site due to remoteness location, necessitating tissue preservation may affect fragment size. This study aimed identify combination four approaches and six methods ensure DNA. A single Peltodoris atromaculata (Nudibranchia) specimen was sliced into ∼30 mg sub‐samples, ensuring consistency across 24 preservation‐extraction combinations triplicates. Samples were either stored 4°C, dried room temperature, flash‐frozen liquid nitrogen, or preserved ethanol −20°C. Afterward, they using five commercially available kits specific extraction, as well a custom protocol. Three aspects quality evaluated: total yield, size distribution, availability amplification. Most yielded optimal results only some three aspects. We identified sequencing: CTAB‐based protocol applied frozen samples, Wizard (Promega) Nanobind (PacBio) both ethanol‐preserved paired with Monarch (NEB) kits. The suitability selected confirmed by PacBio sequencing, producing yield 3.6 Gbp (3.2x estimated coverage). indicate success extractions influenced methods. Although tested on nudibranchs, these findings are highly useful genomic studies other organisms, which need remote locations before being transported laboratory processing.

Language: Английский

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Comprehensive comparison of the third-generation sequencing tools for bacterial 6mA profiling

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: April 28, 2025

DNA N6-methyladenine (6mA) serves as an intrinsic and principal epigenetic marker in prokaryotes, impacting various biological processes. To date, limited advanced sequencing technologies analyzing tools are available for bacterial 6mA. Here, we evaluate eight designed the 6mA identification or de novo methylation detection. This assessment includes Nanopore (R9 R10), Single-Molecule Real-Time (SMRT) Sequencing, cross-reference with 6mA-IP-seq DR-6mA-seq. Our multi-dimensional evaluation report encompasses motif discovery, site-level accuracy, single-molecule outlier detection across six bacteria strains. While most correctly identify motifs, their performance varies at single-base resolution, SMRT Dorado consistently delivering strong performance. study indicates that existing cannot accurately detect low-abundance sites. Additionally, introduce optimized method advancing prediction, which substantially improves of Dorado. Overall, our provides a robust detailed examination computational profiling, highlighting insights further tool enhancement research.

Language: Английский

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Chromosomal evolution, environmental heterogeneity, and migration drive spatial patterns of species richness in Calochortus (Liliaceae)

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(10)

Published: Feb. 23, 2024

We used nuclear genomic data and statistical models to evaluate the ecological evolutionary processes shaping spatial variation in species richness Calochortus (Liliaceae, 74 spp.). occupies diverse habitats western United States Mexico has a center of diversity California Floristic Province, marked by multiple orogenies, winter rainfall, highly divergent climates substrates (including serpentine). sequences 294 low-copy loci produce time-calibrated phylogeny, estimate historical biogeography, test hypotheses regarding drivers present-day patterns number. Speciation coexistence require reproductive isolation divergence, so we examined roles chromosome number, environmental heterogeneity, migration local richness. Six major clades—inhabiting different geographic/climatic areas, often base numbers (n = 6 10)—began diverging from each other ~10.3 Mya. As predicted, number increased significantly with heterogeneity elevation, soil characteristics, serpentine presence. Species is greatest Transverse/Peninsular Ranges where clades overlap, topographic complexity provides conditions over short distances, several physiographic provinces meet allowing immigration clades. Recently diverged sister-species pairs generally have peri-patric distributions, maximum geographic overlap between increases first million years since suggesting that chromosomal evolution, genetic divergence leading gametic or hybrid inviability/sterility, and/or small scales may permit co-occurrence.

Language: Английский

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Exploring Nanopore direct sequencing performance of forensic STRs, SNPs, InDels, and DNA methylation markers in a single assay

Forensic Science International Genetics, Journal Year: 2024, Volume and Issue: 74, P. 103154 - 103154

Published: Oct. 12, 2024

Language: Английский

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A targeted long-read sequencing approach questions the association of OXTR methylation with high-functioning autism

Clinical Epigenetics, Journal Year: 2023, Volume and Issue: 15(1)

Published: Dec. 20, 2023

Abstract Background DNA sequence variation and altered epigenetic regulation of the oxytocin receptor gene (OXTR) have been implicated in autism autistic-like behaviors. While previous studies examined subsegments OXTR, nanopore Cas9-targeted sequencing (nCATS) allows deep characterization entire genes with simultaneous assessment 5-methylcytosine (5mC) modification without need for prior amplification or bisulfite conversion. This pilot study uses an nCATS approach to OXTR its regulatory construct screen 5mC compare results between individuals high-functioning (HFA) neurotypical controls (NC). Methods Using extracted from peripheral blood, (Hg38, chr3: 8750381–8770434, 20,054 base pairs) was analyzed by nCATS. probabilities were calculated visualized across differential methylation analysis performed. Results Twenty adults HFA (10 males, 10 females) 20 age- sex-matched NC (± 5 years) included. There no apparent group differences sequence, except intron variant rs918316, which clustered group. However, did not reveal a single significant group-dependent differentially methylated site among 412 CpG sites captured. Limitations this include small number samples due nature study, particularly limits relevance variants found. It should also be noted that use blood material ability draw conclusions about central processes. Conclusions Previous findings autism-associated alterations reproducible our method. In opinion, may lead reconsideration at individual positions research. given these replicated independent cohorts larger sample sizes.

Language: Английский

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Evaluation of DNA extraction kits for long-read shotgun metagenomics using Oxford Nanopore sequencing for rapid taxonomic and antimicrobial resistance detection

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: Nov. 27, 2024

Abstract During a bacterial infection or colonization, the detection of antimicrobial resistance (AMR) is critical, but slow due to culture-based approaches for clinical and screening samples. Culture-based phenotypic AMR confirmation require up 72 hours (h) even weeks slow-growing bacteria. Direct shotgun metagenomics by long-read sequencing using Oxford Nanopore Technologies (ONT) may reduce time species gene identification. However, swabs complex range Gram-negative -positive bacteria, diverse genes, host DNA present in Therefore, extraction critical initial step. We aimed compare performance different protocols ONT applications reliably identify genes metagenomic approach. included three sample types: ZymoBIOMICS Microbial Community Standard, an in-house mock community ESKAPE pathogens including Enterococcus faecium , Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa Escherichia coli ( Mock ) anonymized swab processed all types with four kits utilizing lysis (enzymatic vs. mechanical) purification (spin-column magnetic beads) methods. used from Qiagen (QIAamp Mini QIAamp PowerFecal Pro DNA) Promega (Maxwell RSC Cultured Cells Maxwell Buccal Swab DNA). After extraction, samples were subject Rapid Barcoding Kit (RBK004) library preparation followed on GridION R9.4.1 flow cells. The fast5 files base called fastq Guppy High Accuracy (HAC) mode inbuilt MinKNOW software. Raw read quality was assessed NanoPlot human reads removed Minimap2 alignment against Hg38 genome. Taxonomy identification performed raw Kraken2 assembled contigs Minimap2. identified CARD database both contigs. Zymo (8/8) (6/6) kit (chemical mechanical lysis) at assembly levels. Enzymatic retrieved fewer aligned bases Gram-positive level compared lysis. detected maximum median 1.9 h sequencing. Long-read turnaround genes. Currently, along captured best taxonomy our specific use case.

Language: Английский

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Emerging methods in botanical DNA/RNA extraction

Applications in Plant Sciences, Journal Year: 2023, Volume and Issue: 11(3)

Published: May 1, 2023

Analyses of nucleic acids (DNA and RNA) have become a staple tool for botanists to answer questions across wide variety disciplines, ranging from population genetics biogeography, ecology, development, microbiology, physiology, phylogenetics. The rise "next-generation" or "high-throughput" sequencing in particular has resulted reduced costs an explosion the number botanical studies using DNA RNA data (Egan et al., 2012). Yet, crucial step extracting these plant tissues can be extremely difficult is often overlooked under-emphasized. Although there are many options acid kits nearly countless papers (over 22,000 at time this special issue) referencing "modified" version Doyle (1987) cetyltrimethylammonium bromide (CTAB) extraction protocol, taxon-specific difficulties render methods ineffective. Troubleshooting remains major sink researchers' energy, potentially acting as barrier downstream analyses answering fundamental questions. Difficulties arise due factors such diversity volume secondary metabolites expressed by plants (Varma 2007), degradation during storage (Pyle Adams, 1989), contamination organisms microbiome (Trivedi 2022), need high-molecular-weight (Pollard 2018). Addressing issues requires knowledge both underlying chemistry involved each process requirements isolated product. 12 issue, "Emerging Methods Botanical DNA/RNA Extraction," highlight current state extractions, including key challenges creative innovations that been developed circumvent address exciting most notable protocol CTAB-based approach (1987). However, researchers commonly refer "modified CTAB" approach, where various modifications highly varied, but without detailing what aspects were adjusted. To better understand alterations, first paper issue (Schenk 2023) reports results literature survey summary modified CTAB protocols. Schenk al. (2023) report provide recommendations eight steps protocol: tissue preparation, suspension, lysis, isolation, cleaning, elution, cleanup, quantification, with explanations why may require modification. Additionally, they four supplementary protocols appendices, which detail alterations lysis and/or steps. This review will allow troubleshoot their own extractions while also promoting repeatability transparency. Many designed "recalcitrant" plants, species whose chemical composition anatomy make much more difficult. Three articles specific ways improve prove recalcitrant different reasons. Recalcitrant contain high amounts hydrolytic enzymes exhibit nuclease activity, do silica-dried tissues. G. Johnson develop demonstrate utility ethanol, rather than silica gel, desiccant prior increase yield quality. Ethanol advantageous it act inhibit enzyme activity easier cell wall disruption. authors compare ethanol three examples: direct collection Vitaceae, pretreatment mangrove Rhizophora mangle (Rhizophoraceae), herbarium specimens 30 taxa representing nine families. increases quality quantity extracted DNA, especially appropriate proteinase digestion other treatments lysis. sclerophylly, Jones investigate effects mechanical disruption, time, leaflet age on concentration thick rigid leaves cycad genus Encephalartos. In cycads, leaf yielded greater concentrations when was manually disrupted mortar pestle vs. bead-based although purity unaffected disruption method. Tissue stored gel concentrations, no impact purity, compared freshly collected tissue. ranged less year decade, significant effect resulting concentration. Finally, reveal senescing young leaflets could adequately sufficient, pure applications. challenging, given presence polyphenols polysaccharides found woody species. Hadi Stacy test effectiveness isolation (along modifications) Metrosideros (Myrtaceae) differ anatomical makeup. They use absorbance ratios, integrity (RIN), success RNA-Seq only one popular kit effective taxa, further optimize high-quality suitable RNA-Seq. optimization extraction. As conduct research field settings, ability rapidly extract great advantage, not store well. Selz polymeric microneedle patches collect directly field. patches, originally drug delivery, produced lab taken After brief application surface, elution ultrapure water cleaning via spin column before ready patch tested standard nuclear chloroplast barcoding loci similar commercial its barcode represented National Center Biotechnology Information's GenBank database. offers flexible practical option remote locations. immediate useful collection, housed long-term provides opportunities historical contemporary existing specimens. Herbarium represent rich resource explore related climate change evolution, well providing access rare even extinct taxa. feasible, needs optimized account changes maximize small so used additional purposes future (Besnard 2018; Funk, Gouker previously published study procedure, species, specimen yield. While outperformed terms all enough subsequent experiments. Interestingly, entire set, (commercial kit, acetone, CTAB) trend within old (before 1960) new (after samples, species-specific differences between existed. Different unique affect acids. Two explicitly methods, Carey experimentally how additives incubation considerations genera. Based results, recommend shorter cooler periods fresh tissue) possible. Recently, moved toward storing frozen samples along typical samples. McAssey creatively set—the Hawaiian Plant Library, 1994–2019—with accessions sheets method A comparison paired revealed obtained significantly fragmented freezer, consequently lower-quality assemblies. Nevertheless, recovery genes short-read high-throughput affected method; instead, had substantial gene recovery. collections continue extraction, adding practice herbaria our evidence biodiversity future. Environmental (eDNA) assess taxonomic present environmental sources, air organismal products (like feces honey), water, soils (M. D. 2023), microbiome, i.e., biotic communities 2022). combined eDNA (eDNA metabarcoding, metagenomics) expanding potential identify (Deiner 2017). developments, technologies describe being applied diverse types. For example, Guillen-Otero ITS 18S rRNA metabarcoding characterize fungal exists ferns lycophytes. conducted relatively challenging tissue—root tissue—to arbuscular mycorrhizal fungi. measured lycophytes important insights into recent common ancestor shared angiosperms. Long-read platforms offer capability generating sequences tens hundreds thousands base pairs. These platforms, companies PacBio (Menlo Park, California, USA) Oxford Nanopore Technologies (Oxford, United Kingdom), sometimes referred "third-generation" technologies. Long reads helped facilitate transition assembly short (50–200 bp length) efficient means whole genome sequencing, 800 genomes sequenced (Marks 2021). Moreover, heavy transposable elements (TEs), long-read technology expand breadth (Shahid Slotkin, 2020). (fragments 50 kbp longer), generally modification obtaining fragment lengths little degradation. end, De La Cerda series straightforward (including beads cut intact pipette tips) nanopore enable evolutionary Calochortus, large genomes. Furthermore, sodium dodecyl sulfate–based promote less-contaminated critical Any length result run, primary concern working clades known having complementary fashion, Kang performed set spanning 18 orders. Testing continues choice studies. combines (to reduce organellar DNA) method, problems associated metabolites. novel longer fragments contamination. Importantly, recovered fraction long reads, variability among presumably varying levels compounds naturally leaves. fern lineages notoriously genomes, necessitates ensure sufficient quantities complete sequencing. Xie presented two yield: strategy prevent shearing nuclei substantially larger DNA. established preventing improves performance (Gong 2019), introduce that, requiring input tissue, generates order magnitude per approaches. (2023), read N50 scores >14 (Wickell 2021; Rahmatpour 2023). Overall, uses questions, cutting-edge generate data, consider all-important concerns, source materials, solutions molecules. We hope help readers protocols, toolkits, frontiers enabled data. N.M. prepared draft manuscript. All provided select article summaries reviewing editing assistance approved final thank submitting work reviewers lending expertise time. Thank you Applications Sciences editor-in-chief Dr. Briana L. Gross managing editor Beth Parada invaluable throughout editorial process.

Language: Английский

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A CRISPR‐based strategy for targeted sequencing in biodiversity science

Molecular Ecology Resources, Journal Year: 2023, Volume and Issue: 24(3)

Published: Dec. 28, 2023

Abstract Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for barcoding and metabarcoding employ PCR amplicon sequencing identify taxa based on target sequences, but target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages some applications. We identified 54,837 guide RNAs that be useful enriching chloroplast across phylogenetically diverse plant species. tested subset 17 vitro enrich strands ranging size barcodes 1,428 bp entire genomes 121,284 bp. an Oxford Nanopore sequencer evaluate success both samples, which yielded mean sequence lengths 2,530–11,367 bp, depending experiment. In comparison experiments, single‐species experiments more on‐target reads greater pairwise identity between contigs species' genomes. But nevertheless, these sufficient data provide ≥48‐fold increase length better estimates relative abundance commercially prepared mixture species compared trn L‐P6 marker. Prior work developed CRISPR‐based protocols long‐read our pioneered its use assemblies have over workflows short‐read sequencing. Future would benefit continuing develop silico analyses especially when appropriate contig assembly cannot known priori.

Language: Английский

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