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Statistical analysis and dataset for: A high-throughput and sensitive method for food preference assays in walking insects
<p>Linked to the journal article published in bioRxiv (https://doi.org/10.1101/2024.04.10.588882).</p> <p><em><strong>Abstract</strong></em></p> <p>Insects pose significant challenges in both pest management and ecological conservation. Often, the most effective strategy is employing toxicant-laced baits, which must also be designed to specifically attract and be preferred by the targeted species for optimal species-specific effectiveness. However, traditional methods for measuring bait preference are either non-comparative, meaning that most animals only ever taste one bait, or suffer from methodological or conceptual limitations. Here we demonstrate the value of direct comparison food preference assays using the invasive and pest ant <em>Linepithema humile </em>as a model. We compare the food preference sensitivity of non-comparative (one visit to a food source) and sequential comparative (visiting one type of food then another) assays at detecting low levels of aversive quinine in sucrose solution. We then introduce and test a novel dual-choice feeder method for simultaneous comparative evaluation of bait preferences, testing its effectiveness in discerning between foods with varying quinine or sucrose levels. While the non-sequential assay could not detect aversion to 1.25mM quinine in 1M sucrose, the sequential comparative approach detected aversion to quinine levels as low as 0.94mM. The novel dual feeder method approach could detect aversion to quinine levels as low as 0.31mM, and also preference for 1M sucrose over 0.75M sucrose. The dual-feeder method, combines the sensitivity of comparative evaluation with high throughput, ease of use, and avoidance of interpretational issues. This innovative approach offers a promising tool for rapid and effective testing of bait solutions, contributing to the development of targeted control strategies. Moreover, the method can be easily modified for application to a wide range of walking insects, such as cockroaches, crickets, and beetles.</p>
Dataset of the paper entitled methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)
<p>Title: Methods for high-throughput screening of novel agents against the maize pest, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) </p> <p>Authors: Sri Ita Tarigan, Gyorgy Turoczi, Jozsef Kiss, Stefan Toepfer</p> <p>Abstract: <br>The western corn rootworm, <em>Diabrotica virgifera virgifera</em> (Coleoptera: Chrysomelidae), poses a significant threat to maize crops in North America and Europe, necessitating development of novel, effective, and less disruptive crop protection agents. With recent bans on key insecticides and concerns about overuse of remaining options, there is an urgent need for accessible and comparable screening methods. We propose comparative high-throughput screening methods against the eggs, larvae and adults of this pest, emphasizing the importance of suitable positive controls tailored to the specific bioassay types. We evaluated seven common insecticides (imidacloprid, clothianidin, acetamiprid, novaluron, cypermethrin, chlorpyrifos-methyl, spinosad) against eggs, larvae, and adults as potential positive controls for each of the proposed assay methods. Dipping assays with ready-to-hatch eggs revealed several ingredients to cause mortality; but imidacloprid might be most suitable as a positive control due to its robust dose-response in reducing egg hatching and causing mortality of hatching neonates. Larval bioassays using artificial diet overlay assays revealed mortality caused by all insecticides, with imidacloprid and acetamiprid exhibiting best dose-mortality response curves as well as sublethal effects. Adult bioassays using artificial diet-core overlay assays revealed mortality caused by all insecticides, with cypermethrin or acetamiprid exhibiting best dose-mortality response curves. The provided ED <sub>50</sub>, ED <sub>80</sub> values, and dose-response equations offer valuable insight for researchers in selecting appropriate positive controls for screening new crop protection agents or assessing resistance levels against different life stages of this pest.</p> <p>Data:</p> <p>The data file is related to the screening of commercial insecticides against eggs, first instar larvae (L1) and adults of the maize pest, <em>Diabrotica virgifera virgifera</em> using standard bioassays. We are proposing comparative high-throughput screening methods against the eggs, larvae and adults of this maize pest. This includes the crucial aspect of suitable positive controls tailored to the specific bioassay type. We evaluated seven common insecticides (imidacloprid, clothianidin, acetamiprid, novaluron, cypermethrin, chlorpyrifos-methyl, spinosad) against eggs, larvae, and adults as potential positive controls for each of the proposed assay method. To access effects and dose-responses of commonly used insecticides on eggs, we applied standard screening methods under controlled semi-sterile conditions.</p> <p>For egg bioassays, eggs were transferred to the 200 ml of treatments in the eppendorf tubes and then soaked for 1 hour. Then 20µl with 10 to 20 eggs were pipetted onto a filter paper in a petri dish (150 mm×25 mm). Then 100 µl of sterilized tap water was added for moisture. The pipette tip was replaced between treatments. The eggs been transferred were counted per filter paper and dish (15± 8). The eggs were then incubated in the dishes at 23-25<sup>0</sup>C for 7 days, when the experiment was terminated. Egg hatching, mortality of newly hatching larvae, and days until start of egg hatching were observed under stereo microscope and recorded. Data were collected at 1,3, 5 and 7 days after treatments.</p> <p>To assess the effect and dose-responses of commonly used insecticides on neonates of <em>D. v. virgifera</em>, we applied artificial diet-overlay bioassays under controlled semi-sterile conditions. Each insecticide was prepared in at least six concentrations. Each bioassay consisted of 3 to 6 polystyrene plates of 96 wells each (07-6096 of Biologix Ltd., USA, or Costar 3917 of Corning Inc., USA). Each well had a volume of 330 µl, with a diameter of 5 mm, a height of 10 mm, and a surface area of 0.34 cm². 190 µl of the diet were pipetted into each 330 µl well, filling each to approximately 2/3<sup>rd </sup>of its capacity. Plates containing the diet were left to dry in a laminar flow cabinet for 45 minutes and then stored overnight at temperatures ranging from 3 to 5°C. The following day, treatments were applied. This is, 17 µl of a treatment was applied to the 0.34 cm<sup>2</sup> diet surface reaching good coverage and therefore forcing the after-placed larvae to feed through (10 to 100 µl pipette Biohit TM Proline). Each treatment was applied to 8 wells per plate. Following application, the plates were allowed to dry for a duration of 1 to 1.5 hours and were subsequently cooled for 1 hour in a refrigerator set at temperatures between 23 to 25°C. Each well received one neonate larva, carefully placed on the diet surface using a fine artist brush. A vigorous and visibly healthy larva was selected, lifted from the end of the abdomen with the brush, maneuvered towards a well surface, and allowed to crawl off the brush onto the diet. To avoid systematic errors, larvae were not arranged in treatment column order but rather in a rectangular pattern. After every 12 individual larvae, the brush was cleaned using 70% ethanol followed by sterile tap water. The filled plate was sealed with an optically clear adhesive qPCR seal sheet (#AB-1170, Termo Scientific, USA, or #BS3017000, Bioleader, USA), enabling data assessments without the need to open the plate. Four to five holes were carefully made with fine 00-insect pins into the seal per well to facilitate aeration. The plates, housing the larvae, were then incubated in a dark, ventilated incubator at a temperature of 23-25 °C and a relative humidity of 50 to 90% for a period of 5 days. We assessed mortality and stunting larvae within 3 and 5 days. </p> <p>To access the effect and dose-responses of common insecticides on <em>D.v.virgifera</em> adults, artificial diet-overlay bioassays with different doses were performed under controlled, semi-sterile conditions. Each insecticide was prepared in at least six concentrations. Active ingredients as specified on the product labels underwent serial dilutions using sterile tap water. Sterilized tap water was used as untreated control. In detail, each bioassay consisted of 6 polystyrene plates of 6 wells each (Eppendorf® 0030720016). Each treatment was applied to 3 wells of each plate per bioassay. The adult diet for a bioassay had been prepared 1-7days before treatment and adult infestation. The diet was prepared under semi-sterile conditions. The diet was poured out to 5-6 sterile 11 mm Petri dishes. The plates with diet were allowed to dry for up to 15 minutes under laminar flow cabinet then stored at 3 to 5°C overnight.The following day, a core of the diet was initially transferred to each well using flamed iron core-cutter (1 cm diameter) under a laminar flow. A core diet was placed each of the 6 wells of the plates. Approximately 40 µl of the treatments were then applied across the surface of diet core (0.34 cm<sup>3</sup>). The following day, a core of the diet was initially transferred to each well using flamed iron core-cutter (1 cm diameter) under a laminar flow. A core diet was placed each of the 6 wells of the plates. Approximately 40 µl of the treatments were then applied across the surface of diet core (0.34 cm<sup>3</sup>). Adult were subsequently transferred from the rearing cage into the wells of the 6-well plates containing the diet and treatments using a tube aspirator. For ease of transfer, the adults were cooled in a fridge for 4 to 7 minutes. Each well plate received 3 to 4 adults. Plates were sealed and incubated at 23-25<sup>0</sup>C, 50–90% r.h, L: D 12:12. Adult mortality were recorded on days 1,3, 5, 7 of experiment. </p> <p>To allow comparisons between experiments, data were standardized to the data of the corresponding negative control, usually sterilized tap water, as follows: standardized data = 100 × (data in negative control - data in treatment)/maximum (data in control or in treatment). The distributions of the data were investigated using histograms and QQ normal and detrended normal probability. Skewness and kurtosis of residuals was also observed for normality of influences of treatments on eggs, neonates, or adults. Equality of variances was assessed using Levene’s test. Multiple comparisons were performed using the Tukey HSD post hoc test for data with equal variances and the Games-Howell post hoc test for data with unequal variances. For each tested insecticide, linear and logarithmic regression models were fit to the dose-response data. In case of significant linear or logartimic relathionships, doses leading to 50% or 80% of relative effects (ED <sub>50,80</sub>) were calculated. </p> <p>The raw data as well as the standardised data are available as a csv file on zenodo. </p> <p> </p> <p> </p>
TREXIO files used for the validation tests in the paper entitled 'TurboGenius: Python suite for high-throughput calculations of ab initio quantum Monte Carlo methods'.
<p>The TREXIO files used for the validation tests in the paper entitled TurboGenius: Python suite for high-throughput calculations of ab initio quantum Monte Carlo methods. The detail about the TREXIO library is described in the JCP article [J. Chem. Phys. 158, 174801 (2023)] and the GitHub repository [https://github.com/TREX-CoE/trexio]. The TREXIO files were generated using TREXIO version 2.3.2 (and the corresponding Python API version 1.3.2).</p>
Data for: "A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death"
<p>Data related to the publication Murschhauser <em>et al.</em>: <a href="https://doi.org/10.1038/s42003-019-0282-0">A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death</a>. It contains fluorescence time traces of single cells marked with cell-event markers and observed by time-lapse microscopy. The cells were treated with nanoparticles at different doses (NP25 and NP100), with staurosporine (sts) or were left untreated for control (ctrl). See the above-mentioned publication for more details.</p> <p>The format of the data is described below.</p> <p>The file <code>Data_A549.zip</code> contains data measured with A549 cells, and the file <code>Data_Huh7.zip</code> contains data measured with Huh7 cells. Both files have the same structure. Each file contains the directories <code>Raw</code> and <code>Fitted</code> as well as a checksum file. The <code>Raw</code> directory contains single-cell fluorescence time courses as obtained by time-lapse microscopy. The <code>Fitted</code> directory contains the results of fitting model functions as well as properties of identified events, such as event times. The checksum file contains SHA256 checksums of all files within these directories and can be used to check file integrity.</p> <p>Both directories contain measurement directories. Each measurement directory contains the data corresponding to one experiment. The name of the measurement directory is the measurement identifier. Each measurement directory contains condition directories. Each condition directory contains data corresponding to one condition measured in the measurement and is named after the condition. Each condition directory contains marker directories. They are named after the fluorescence markers measured and contain files with single-cell data corresponding to the respective markers.</p> <p>The names of those files consist of multiple parts separated by underscores. The first two parts identify a position of the microscope. Since pairs of markers were measured, each position is present in two marker directories. The third part is the measurement identifier. The other parts will be described below.</p> <p>The <code>Raw</code> directory contains only CSV files with the raw fluorescence time courses. The filenames contain no other parts and have the suffix “.txt”. The first row of each CSV file is the time (in units of 10 minutes), and the other rows are the fluorescence time courses of the cells observed at the corresponding position (in arbitrary units). Each file in the <code>Raw</code> directory corresponds to a group of files in the <code>Fitted</code> directory.</p> <p>The <code>Fitted</code> directory contains three types of CSV files. Their names have “ALL” as fourth part, a session identifier as sixth part and the suffix “.csv”. The fifth part indicates the type of file and is one of the following:</p> <ul> <li>“PARAMS” indicates the estimated values for the model parameters. Each row stands for one cell and each column for a parameter of the model function fitted to the data. The model functions are published with the <a href="https://doi.org/10.5281/zenodo.1418465">fitting software</a>.</li> <li>“SIMULATED” indicates the fitted traces. The traces are calculated using the model functions and the estimated parameters. The format is the same as for the raw traces, but the time is in units of hours and has a higher resolution.</li> <li>“STATE” indicates additional information extracted from the fitted traces. Each row stands for a cell and each column for a property. The first column is the number of the cell. The second column is the event time found (in hours); non-finite values indicate that no event time was found. The third and fourth columns contain the absolute and relative amplitude of the trace, respectively. The fifth column is the logarithmic likelihood of the best fit. The sixth column indicates an algorithm used for postprocessing, and the seventh column indicates the trace slope at the event. See the fitting software for details.</li> </ul> <p> </p>
Figure S1 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure S1. Map of the Gigantometra gigas mitogenome using Sanger method (GenBank accession number: MF177288). Genes in the outer circle indicate the direction of transcription of the majority strand (J-strand), and those in the inner circle indicate that of the minority strand (N-strand). The GC content, GC skew+, and GC skew- are separately shown in the circle.
Figure 6 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 6. Two examples of the heteroplasmic sites in Sanger sequencing which correspond to the differently sequenced sites. Panels A and B indicate the sites at which the second-peak is obviously higher than the third-peak and fourth-peak, and the base state of the second-peak can be obtained by at least one result of HTS. The different fluorescence densities of base situated at np 1923 in the cox1 are shown in the panel A, and the panel B shows the nucleotides with amino acids at np 1923 in the results of Sanger and HTS methods. The nucleotides are C in the results of HTS sequencing, while the corresponding nucleotides are T in the results of Sanger method in both positions, and the different nucleotides lead not to the amino acids changed. Panels C and D indicate the site at the unobvious second-peak, which is slightly higher than the third-peak and fourth-peak, and the base state of the second-peak can also be obtained by at least one result of HTS. Panel C shows the unobvious second-peak at np 7125, and the nucleotide and amino acid of the site in the results of Sanger and HTS methods are shown in panel D. The amino acids are listed using single-letter amino acid abbreviations.
Figure 4. Intraspecific pairwise K2P in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 4. Intraspecific pairwise K2P distance of G. gigas based on barcode fragment size of cox1 (Sanger). The red boxplot shows the genetic distances of individuals in all three collecting sites, and the boxplots (blue, green, and yellow) separately show the distances of individuals within each place (HNYG, HNDL, and VIET). The pink boxplot shows the distances of the corresponding cox1 sequences obtained by the two sequencing methods. Abbreviation: HNYG—Yinggeling Nature Reserve, Hainan; HNDL— Diaoluoshan Nature Reserve, Hainan; VIET—northern Vietnam.
Figure S5 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure S5. The coverage of short fragments at each position in the assembly results of HTS. The three results of HTS method were separately used as reference sequences to be mapped back onto the corresponding HTS scaffolds, and the mitochondrial genes were shown below the corresponding coverage. The scale bar had an indicator at the mean coverage level and the coverage for each nucleotide position was indicated by the height of the blue line.
Figure 3 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 3. The different nucleotides in the ITS-1 and ITS-2 regions are shown. The result shows the different nucleotides at nucleotide position np 1897 (G nucleotide and T nucleotide) and np 2790 (C nucleotide and T nucleotide) obtained by Sanger and HTS methods.
Figure 1. Gigantometra gigas. A. Female, dorsal view. B. Male, dorsal view. C in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 1. Gigantometra gigas. A. Female, dorsal view. B. Male, dorsal view. C. The narrow distribution of G. gigas.
Data for: Tools and methods for high-throughput single-cell imaging with the mother machine
Open the record for dataset details and reuse information.
Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?
<p>The 8 benthic samples from Mainland France (stream Edian, stream Aire, lake Geneva), Sweden (stream Dåmman, Agricultural stream, lake Båtkåjaure) and Mayotte (stream Dapani, stream Majimbini) were collected by scraping material from the surface of stones, following the French standard (AFNOR 2007) used in routine biomonitoring programs.DNA was extracted from each sample (2 replicates) using five DNA extraction methods, followed by the amplification of a short rbcL DNA barcode (312bp) specific to diatoms. PCR products were then sequenced in one random direction using the Ion Torrent™ Personal Genome Machine® (PGM) System according to the manufacturer’s instructions. The data file contains one fastq file per library sequenced with the raw DNA reads, as provided by the sequencing platform (demultiplexing performed by the sequencing platform). An excel file is also provided to make the link between the fastq file number and the sample information (sample origin, DNA extraction method used, number of raw reads).</p>
ZymoSoups: A high-throughput forward genetics method for rapid identification of virulence genes in Zymoseptoria tritici
<p>These data were used to generate figures and summarise the number of mutations in 18 re-sequenced genomes of <em>Zymoseptoria tritici. </em>The isolates genotyped in this VCF file are all UV-mutegenized strains of the reference isolate IPO323.</p> <p><strong>Abstract</strong></p> <p><span><span>Septoria tritici blotch is caused by the fungus </span></span><em><span><span>Zymoseptoria</span><span> tritici</span> </span></em><span><span>and</span><span> poses a major threat to wheat </span><span>productivity</span><span>. </span><span>There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance </span><span>against this pathogen</span><span>, h</span><span>owever</span> <span>t</span><span>he corresponding genes in </span></span><em><span><span>Z. tritici</span></span></em><span><span> that confer virulence against distinct </span></span><span><span>R</span></span><span> <span>genes </span><span>remain</span> <span>largely unknown</span><span>. </span><span>In this study, we developed a</span> <span>rapid </span><span>forward genetics </span><span>methodology</span> <span>to </span><span>identify</span><span> genes that enable </span></span><em><span><span>Z. tritici</span></span></em><span><span> to gain virulence </span><span>on </span><span>previously resistant wheat varieties. </span><span>We</span><span> used the known</span><span> gene-for-gene interaction between </span></span><span><span>Stb6</span></span><span><span> and </span></span><span><span>AvrStb6</span></span><span><span> as </span><span>a </span><span>proof of concept that this method could quickly recover single candidat</span><span>e</span><span> virulence genes</span><span>. We subjected the avirulent </span></span><em><span><span>Z. tritici</span></span></em><span><span><em> </em>strain IPO323</span><span>, which carries the recogni</span><span>z</span><span>ed </span></span><span><span>AvrStb6</span></span><span><span> allele,</span><span> to UV mutagenesis</span><span> and</span><span> generat</span><span>ed</span><span> a library of over </span><span>66</span><span>,</span><span>000 </span><span>surviving spores</span><span>. </span><span>We screen</span><span>ed</span><span> these </span><span>survivors</span><span> on leaves of the resistant wheat variety Cadenza</span><span>,</span><span> in</span><span> mixtures</span> <span>(</span><span>soups</span><span>)</span><span> ranging from </span><span>100</span><span>-</span><span>500</span> <span>survivors</span><span> per soup. </span><span>We </span><span>identified</span><span> five soups</span><span> with </span><span>a </span><span>gain</span><span>-</span><span>of</span><span>-</span><span>virulence (</span><span>GoV</span><span>)</span><span> phenotype </span><span>relative</span><span> to the IPO323 parental strain</span><span> and re-sequenced 18 individual isolates, including four control isolates and two </span><span>isolates</span><span> lacking virulence</span><span>,</span><span> when screened </span><span>individually.</span> <span>Of </span><span>the </span><span>12 </span><span>confirmed </span><span>GoV</span> <span>isolates</span><span>, o</span><span>ne had a single nucleotide polymorphism (SNP) in the </span></span><span><span>AvrStb6</span></span><span><span> coding region. </span><span>T</span><span>he other 11 </span><span>GoV</span> <span>isolates</span> <span>exhibited</span> <span>large</span><span> (~70Kb)</span> <span>deletions at the end of chromosome 5, including the </span></span><span><span>AvrStb6</span></span><span><span> locus. </span><span>Our findings </span><span>demonstrate</span><span> the </span><span>efficiency </span><span>of this forward genetic approach in elucidating the genetic basis of qualitative resistance to </span></span><span><span>Z. tritici</span></span><span><span> and the </span><span>potential </span><span>to rapidly </span><span>identify</span><span> other</span><span>,</span><span> currently unknown</span><span>,</span> </span><span><span>Avr</span></span><span><span> genes in this pathogen. </span></span></p> <p><span><strong>Analysis pipeline</strong></span></p> <p><span>The pipeline used to generate this filtered vcf file and tab delimited file is publicly available at https://github.com/megancamilla/GATK4_Zymoseptoria</span></p> <p><span><strong>Isolate Data Accessibility</strong></span></p> <p><span><span>The individual fastq files for each isolate included in this study have been uploaded to NCBI's Short Read Archive (SRA), project number PRJNA1017776. This project </span><span>contains</span><span> 18 re-sequenced samples (SAMN37407989-SAMN37407972),</span> <span>which are UV mutagenized IPO323. Short Read Archive (SRA) accession numbers are SRR26072101-SRR26072118. </span> </span></p>
Supplementary materials for "High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method."
<p>The next-generation sequencing (NGS) data of the 5th rounds' samples for LX9 library and p53deg library. The csv files contain DNA sequences read out, amino acid sequences and their read counts in descending order. </p>
Data from: A high-throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia
Open the record for dataset details and reuse information.
Supporting Information: High-throughput Saccharomyces cerevisiae cultivation method for credentialing-based untargeted metabolomics
<p>F1: Physiological Constraints: Growth rate, glucose uptake, and intracellular 13C succinate concentration</p> <p>F2: PAVE input and adduct list</p> <p>F3: Credentialing results, HILIC data</p> <p>F4: Credentialing results, RP lipids data</p> <p>F5: HILIC Level 2A annotation with MS-DIAL and manual mass shift quality control</p> <p>F6: R script for the MetFrag/PCLite-based annotation</p> <p>F7: HILIC annotation with MetFrag/PCLite</p> <p>F8: RP-lipids annotation with MS-DIAL</p> <p>F9: Level 1 compounds identification</p> <p>F10: InChKey-based YMDB and HMDB recovery analysis </p> <p>F11: InChKey-based comparison with PAVE publication</p> <p>F12: Statistic Results</p> <p>F13: Supernatant measurements</p>
Data from: Sequence Capture using PCR-generated Probes (SCPP): a cost-effective method of targeted high-throughput sequencing for non-model organisms
Recent advances in high-throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of non-model organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in-solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR-generated probes (SCPP) protocol requires no specialized equipment, is highly flexible, and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25-100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.
A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation
<p></p><p>Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10−8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10−4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10−5 to 10−1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive base read was evaluated to determine if there is a limit of read length for quantitation of ≥10−4, and it was determined that read lengths up to 70 bases are reliable for quantitation. The method described here is adaptable to various oncogene or tumor suppressor gene targets, with the potential to implement multiplexing at the initial tagging step. While easy to perform manually, it is also suited for robotic handling and batch processing of samples, facilitating detection and quantitation of genetic carcinogenic biomarkers before tumor formation or in normal-appearing tissue.</p><p></p>
Figure 5. The polymorphism sites among the 25 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 5. The polymorphism sites among the 25 different cloning sequences of cox1. Weblogo 3.0 was used to show the nucleotide content of 25 cloning sequences of cox1 (Crooks et al., 2004). The abscissa stands for the number of the bases, while the ordinate stands for the proportion of nucleotide content provided by the 25 different cloning sequences in the same position. The sequence length between the two arrows stands for the barcode fragment size of cox1. The black triangles show the polymorphism positions in the 25 different cloning sequences, the red circles show the positions exhibited obvious second-peak in the results of direct Sanger sequencing without cloning, and the yellow stars show the different sites between the results of Sanger and HTS.
Figure 2 in Biased heteroplasmy within the mitogenomic sequences of Gigantometra gigas revealed by sanger and high-throughput methods
Figure 2. The different nucleotides of all 13 PCGs in mitogenomes obtained by Sanger and HTS sequencing. The different sequences of HTS sequencing are separately compared with the consequence of Sanger method. The horizontal axis stands for the nucleotide position, of which the sequences of 13 PCGs are ordered according to the circular mitochondrial DNA from nad2 to nad1 in the clockwise direction. The vertical axis stands for the number of the different sites in the sequences of 13 PCGs, and the different nucleotide in each site of all three HTS sequences compared with Sanger is shown in the corresponding panel.
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.