Ringraziamo Taylor AuBuchon-Elder, Jordan Teisher e Kristina Zudock per assistenza negli esemplari di erbario di campionamento e il giardino botanico del Missouri per l'accesso agli esemplari di erbario per campionamento distruttivo. Questo lavoro è stato supporto da una sovvenzione della National Science Foundation (DEB-1457748).

<ol>
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Il protocollo presentato qui è un metodo completo e robusto per isolamento del DNA e sequenziamento preparazione libreria dagli esemplari di piante essiccate. La consistenza del metodo e minima necessità di modificarla basata su esemplare qualità rendono esso scalabile per progetti di grande erbario di sequenziamento. L'inclusione di un passo opzionale reamplification per librerie di basso rendimento permette l'inclusione di bassa qualità, quantità di basso, rara o storicamente importanti campioni che altrimenti non...

Erbario collezioni sono una fonte potenzialmente importante di entrambe le specie e diversità genomica per studi compreso phylogenetics1,2,3, genetica delle popolazioni4,5, conservazione biologia6, specie invasive biologia7e tratto evolution8. La capacità di ottenere una ricca diversità di specie, popolazioni, luoghi geografici e punti di tempo mette in evidenza il "forziere"9 che è l'erbario. Storicamente, la natura degradata del DNA erbario-derivato ha ostacolato progetti basati su PCR, relegando spesso i ricercatori a usando solo gli indicatori trovati in alta copia, quali le regioni del genoma del cloroplasto o il distanziatore interno trascritto (ITS) della ribosomiale RNA. Qualità dei campioni e DNA variano ampiamente basata su metodi di conservazione9,10, con rotture a doppio filamento e la frammentazione da calore utilizzato nel processo di essiccazione, essendo le forme più comuni di danno, creando il cosiddetto 90% DNA di lock-up che ha gravato studi basati su PCR11. A parte la frammentazione, il problema secondo più prevalente in erbario genomica è contaminazione, come quello derivato da funghi endofiti13 o funghi acquisito post mortem dopo la raccolta, ma prima di montare nell'erbario12, però Questo problema può essere risolto bioinformatically dato il database proprio fungo (Vedi sotto). Un terzo problema, meno comune, è modifica di sequenza attraverso citosina deaminazione (C/G→T/A)14, anche se si stima essere bassa (~ 0,03%) in campioni di erbario11. Con l'avvento di sequenziatori di alto-rendimento (HTS), il problema di frammentazione può essere superato con brevi letture e sequenziamento profondità12,15, permettendo l'acquisizione di dati a livello genomico dalle numerosi esemplari con bassa qualità DNA e a volte anche permettendo di sequenziamento del genoma intero15.
I campioni di erbario stanno diventando sempre più frequentemente utilizzati e una maggiore componente di filogenetica progetti16. Una sfida attuale della utilizzando campioni di erbario per HTS è costantemente ottenere sufficiente DNA a doppia elica, un prerequisito indispensabile per protocolli di sequenziamento, da numerose specie in modo tempestivo, senza la necessità di ottimizzare i metodi per individuo esemplari. In questa carta, un protocollo per l'estrazione di DNA e preparazione di libreria di campioni di erbario è dimostrato che si avvale dei metodi esistenti e li modifica per consentire risultati veloci e replicabili. Questo metodo consente completo elaborazione da esemplare a una libreria di 24 campioni in 13 h, con tempo di preparazione manuale h 8, o 16 h, con hands-on tempo h 9, quando è richiesto il passaggio opzionale reamplification. Elaborazione simultanea di più campioni è realizzabile, anche se il fattore limitante è la capacità della centrifuga e abilità tecnica. Il protocollo è progettato per richiedere solo attrezzature tipici del laboratorio (termociclatore, centrifuga e supporti magnetici) invece di attrezzature specializzate, come un nebulizzatore o un sonicatore, per la tosatura del DNA.
Qualità del DNA, la dimensione del frammento e la quantità sono fattori per l'utilizzo di campioni di erbario a esperimenti di sequenziamento ad alta velocità limitanti. Altri metodi per isolare il DNA di erbario e creazione di librerie di sequenziamento ad alte prestazioni hanno dimostrato l'utilità dell'utilizzo di appena 10 ng di DNA16; Tuttavia hanno bisogno di determinare sperimentalmente il numero ottimale di PCR cicli necessari per la preparazione di biblioteca. Questo diventa impraticabile quando trattare con estremamente piccole quantità di vitali double stranded DNA (dsDNA), come alcuni esemplari di erbario producono solo abbastanza DNA per una preparazione unica libreria. Il metodo presentato qui utilizza un singolo numero di cicli indipendentemente dalla qualità del campione, quindi nessun DNA si perde nelle fasi di ottimizzazione biblioteca. Invece, un passo di reamplification viene richiamato quando le librerie non soddisfano gli importi minimi necessari per la sequenza. Molti campioni di erbario sono rari e possiedono poco materiale che lo rende difficile da giustificare campionamento distruttivo in molti casi. Per contrastare ciò, il protocollo presentato permette di dsDNA ingresso taglie meno di 1,25 ng nel processo di preparazione di biblioteca, ampliando l'ambito dei campioni praticabili per high throughput sequenziamento e riducendo al minimo la necessità di campionamento distruttivo degli esemplari.
Il seguente protocollo è stato ottimizzato per erbe e testato su centinaia di specie diverse dai campioni di erbario, anche se ci aspettiamo che il protocollo può essere applicato a molti altri gruppi di piante. Esso comprende un passaggio di ripristino opzionale che può essere utilizzato per salvare bassa qualità e/o rari esemplari. Questo protocollo basato su oltre duecento esemplari di erbario testati, funziona sugli esemplari con tessuto basso input e qualità, consentendo la conservazione di esemplari rari tramite campionamento distruttivo minimo. Qui è indicato che questo protocollo può fornire librerie di alta qualità che possono essere ordinati in sequenza per progetti basati su phylogenomics.

<table><tbody><tr><td>Veriti Thermal Cycler</td><td>Applied Biosystems</td><td>4452300</td><td>96 well&amp;nbsp;</td></tr><tr><td>Gel Imaging System</td><td>Azure Biosystems</td><td>c300</td><td></td></tr><tr><td>Microfuge 20 Series</td><td>Beckman Coulter</td><td>B30137</td><td></td></tr><tr><td>Digital Dry Bath</td><td>Benchmark Scientific</td><td>BSH1001</td><td></td></tr><tr><td>Electrophoresis System</td><td>EasyCast</td><td>B2</td><td></td></tr><tr><td>PURELAB flex 2 (Ultra pure water)</td><td>ELGA&amp;nbsp;</td><td>89204-092</td><td></td></tr><tr><td>DNA LoBind Tube&amp;nbsp;</td><td>Eppendorf</td><td>30108078</td><td>2 ml</td></tr><tr><td>Mini centrifuge</td><td>Fisher Scientific</td><td>12-006-901</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Mortar</td><td>Fisher Scientific</td><td>S02591</td><td>porcelain</td></tr><tr><td>Pestle</td><td>fisher Scientific</td><td>S02595</td><td>porcelain</td></tr><tr><td>Centrifuge tubes</td><td>fisher Scientific</td><td>21-403-161</td><td></td></tr><tr><td>Microwave</td><td>Kenmore</td><td>405.7309231</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>0.2 ml Strip tube and Cap for PCR</td><td>VWR</td><td>20170-004</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>PM2000</td><td></td></tr><tr><td>Liquid Nitrogen Short-term Storage</td><td>Nalgene</td><td>F9401</td><td></td></tr><tr><td>Magnetic-Ring Stand&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>AM10050</td><td>96 well&amp;nbsp;</td></tr><tr><td>Water Bath</td><td>VWR</td><td>89032-210</td><td></td></tr><tr><td>Hot Plate Stirrers</td><td>VWR</td><td>97042-754</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Airgas</td><td>UN1977</td><td></td></tr><tr><td>1 X TE Buffer</td><td>Ambion</td><td>AM9849</td><td>pH 8.0</td></tr><tr><td>CTAB</td><td>AMRESCO</td><td>0833-500G</td><td></td></tr><tr><td>2-MERCAPTOETHANOL</td><td>AMRESCO</td><td>0482-200ML</td><td></td></tr><tr><td>Ribonuclease A</td><td>AMRESCO</td><td>E866-5ML</td><td>10 mg/ml solution</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63882</td><td></td></tr><tr><td>Sodium Chloride</td><td>bio WORLD</td><td>705744</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>bio WORLD</td><td>40970004-1</td><td></td></tr><tr><td>Nuclease Free water</td><td>bio WORLD</td><td>42300012-2</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Fisher Scientific</td><td>A393-500</td><td></td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Fisher Scientific</td><td>s608-500</td><td></td></tr><tr><td>LE Agarose</td><td>GeneMate</td><td>E-3120-500</td><td></td></tr><tr><td>100bp PLUS DNA Ladder</td><td>Gold Biotechnology</td><td>D003-500</td><td></td></tr><tr><td>EDTA, Disodium Salt</td><td>IBI Scientific</td><td>IB70182</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Life Technologies</td><td>Q32854</td><td></td></tr><tr><td>TRIS</td><td>MP Biomedicals</td><td>103133</td><td>ultra pure</td></tr><tr><td>Gel Loading Dye Purple (6 X)</td><td>New England BioLabs</td><td>B7024S</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England BioLabs</td><td>M0348L</td><td></td></tr><tr><td>NEBNext Ultra II DNA Library Prep Kit for Illumina&amp;nbsp;</td><td>New England BioLabs</td><td>E7645L</td><td></td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>New England BioLabs</td><td>E7600S</td><td>Dual Index Primers Set 1</td></tr><tr><td>NEBNext Q5 Hot Start HiFi PCR Master Mix</td><td>New England BioLabs</td><td>M0543L</td><td></td></tr><tr><td>Mag-Bind RXNPure Plus</td><td>Omega bio-tek</td><td>M1386-02</td><td></td></tr><tr><td>GelRed 10000 X</td><td>Pheonix Research</td><td>41003-1</td><td></td></tr><tr><td>Phenol solution</td><td>SIGMA Life Science</td><td>P4557-400ml</td><td></td></tr><tr><td>PVP40</td><td>SIGMA-Aldrich</td><td>PVP40-50G</td><td></td></tr><tr><td>Chloroform</td><td>VWR</td><td>EM8.22265.2500</td><td></td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1016</td><td>200 Proof</td></tr><tr><td>Silica sand</td><td>VWR</td><td>14808-60-7</td><td></td></tr><tr><td>Reamplification primers</td><td>Integrated DNA Technologies</td><td>see text</td><td></td></tr><tr><td>Sequencher v.5.0.1</td><td>GeneCodes</td><td></td><td></td></tr></tbody></table>

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

Gli erbari sono una preziosa fonte di materiale vegetale che può essere utilizzato in una varietà di studi biologici. L'uso di campioni di erbario è associato con una serie di sfide tra cui qualità di conservazione del campione, DNA degradato e campionamento distruttivo di rari esemplari. Per poter utilizzare in modo più efficace materiale di erbario in progetti di sequenziamento di grandi dimensioni, è necessario un metodo affidabile e scalabile di preparazione di isolamento e libreria di DNA. Questa carta dimostra un protocollo robusto, inizio-to-end per DNA alto-rendimento e isolamento costruzione della libreria da esemplari che non richiedono la modifica di singoli campioni. Questo protocollo è su misura per la bassa qualità secchi pianta materiale e approfitta dei metodi esistenti ottimizzando tessuto rettifica, modifica selezione dimensione biblioteca ed introducendo un passo opzionale reamplification per librerie di basso rendimento. Reamplification di librerie di basso rendimento del DNA può salvare i campioni provenienti da campioni di erbario potenzialmente preziosi e insostituibili, negando la necessità per ulteriore campionamento distruttivo e senza introdurre bias di sequenziamento discernibile per comune applicazioni filogenetiche. Il protocollo è stato testato su centinaia di specie di erba, ma dovrebbe essere adatto per l'impiego in altri lignaggi pianta dopo la verifica. Questo protocollo può essere limitato dal DNA estremamente degradato, dove non esistono frammenti nel campo dimensione desiderata, e di metaboliti secondari presenti in alcuni materiale vegetale che inibiscono pulito isolamento del DNA. Nel complesso, questo protocollo introduce un metodo veloce e completo che permette di isolamento del DNA e preparazione della biblioteca di 24 campioni in meno di 13 ore, con solo 8 h di tempo attivo hands-on con modifiche minime.

Isolamento del DNA e la resa finale biblioteca In questo studio, l'efficacia del protocollo per l'isolamento del DNA di erbario e al recupero delle librerie di sequenziamento di alta qualità è stata dimostrata utilizzando cinquanta diversi campioni con la più antica dal 1920 e il più giovane dal 2012 (tabella 2). Per ogni campione, circa 10 mg di tessuto fogliare è stato usato per isolamento del DNA. Più verde tessuto fogliare è stato favorito s...

Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Isolamento del DNA robusto e costruzione della libreria High-throughput sequenziamento per campioni di erbario

Questo articolo viene illustrato un protocollo dettagliato per isolamento di DNA e la costruzione della libreria sequenziamento ad alte prestazioni da materiale di erbario tra cui salvataggio di eccezionalmente scarsa qualità del DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

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JoVE Journal

Genetics

10.5K Views.  Donald Danforth Plant Science Center. Questo articolo viene illustrato un protocollo dettagliato per isolamento di DNA e la costruzione della libreria sequenziamento ad alte prestazioni da materiale di erbario tra cui salvataggio di eccezionalmente scarsa qualità del DNA.

Video: Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Automated Gel Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

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Ambiente

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

Purificazione di alto peso molecolare genomico da oidio per lungo-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

Genetica

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Ottimizzazione e analisi comparativa dei metodi di arricchimento del DNA organologico vegetale adatti per la sequenziazione di nuova generazione

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Empirica, metagenomica e tecniche computazionali illuminano i meccanismi attraverso i quali salute delle API di fungicidi compromesso

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Bassissimo ingresso il sequenziamento del genoma libreria preparazione da un singolo esemplare di Tardigrade

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology. Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to error-prone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination.

Assemblaggio di genoma ibrido De Novo per la generazione di genomi completi di batteri urinari utilizzando tecnologie di sequenziamento a lettura breve e lunga

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data ...

<meta charset="utf8"></head><body>DNA Barcoding per piante medicinali: identificazione e analisi filogenetica

Application of DNA Barcoding to Identify Medicinal Plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

<meta charset="utf8"></head><body>Autore in primo piano: Sfruttare la tecnologia dei codici a barre del DNA per migliorare l'efficienza dell'identificazione delle piante medicinali

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

Applicazione del DNA Barcoding per identificare le piante medicinali

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Gel-seq: Un metodo per la preparazione di libreria simultanea di sequenziamento del DNA e RNA utilizzando matrici di idrogel

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Isolamento del DNA robusto e costruzione della libreria High-throughput sequenziamento per campioni di erbario

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Capitoli in questo video

Automated Gel Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

Purificazione di alto peso molecolare genomico da oidio per lungo-Read Sequencing

Ottimizzazione e analisi comparativa dei metodi di arricchimento del DNA organologico vegetale adatti per la sequenziazione di nuova generazione

Empirica, metagenomica e tecniche computazionali illuminano i meccanismi attraverso i quali salute delle API di fungicidi compromesso

Bassissimo ingresso il sequenziamento del genoma libreria preparazione da un singolo esemplare di Tardigrade

Assemblaggio di genoma ibrido De Novo per la generazione di genomi completi di batteri urinari utilizzando tecnologie di sequenziamento a lettura breve e lunga

Applicazione del DNA Barcoding per identificare le piante medicinali

Applicazione del DNA Barcoding per identificare le piante medicinali

Applicazione del DNA Barcoding per identificare le piante medicinali

Gel-seq: Un metodo per la preparazione di libreria simultanea di sequenziamento del DNA e RNA utilizzando matrici di idrogel