Wir danken Taylor AuBuchon-Holunder, Jordan Teisher und Kristina Zudock um Hilfe bei der Probenahme Herbar und Missouri Botanical Garden Zugang zum Herbar für destruktive Probenahme. Diese Arbeit wurde unterstützt durch einen Zuschuss von der National Science Foundation (DEB-1457748).

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Die Autoren erklären, dass sie keine konkurrierenden Interessen haben.

Die hier vorgestellten Protokoll ist eine umfassende und solide Methode zur DNA-Isolierung und Sequenzierung Bibliothek Vorbereitung von getrockneten Pflanzen. Die Konsistenz des Verfahrens und des minimalen Notwendigkeit, es zu ändern basierend auf Probe Qualität machen es skalierbar für große Herbarium-basierte Sequenzierung Projekte. Die Einbeziehung eines optionalen Reamplification Schritts für geringe Ausbeute Bibliotheken ermöglicht die Aufnahme von geringer Qualität, geringe Menge, selten, oder historisch w...

Herbarium Sammlungen sind potentiell wertvolle Arten und genomische Diversität für Studien einschließlich Phylogenetik1,2,3, Populationsgenetik4,5, Erhaltung Biologie6, invasive Arten Biologie7und Merkmal Evolution8. Die Fähigkeit, eine große Vielfalt von Arten, Populationen, geografische Orte und Zeitpunkte zu erhalten unterstreicht die "Schatztruhe"9 , die das Herbarium. Historisch, hat degradierten Artder Herbarium abgeleitet DNA PCR-basierten Projekten, oft verwies Forscher mittels nur Marker gefunden in hohe Kopie, wie Regionen von Chloroplast Genom oder die interne transkribierten Distanzstück (ITS) aus der ribosomalen behindert. RNA. Qualität der Proben und DNA variieren ausgiebig basierend auf Methoden der Konservierung9,10, mit Doppelstrang-Brüche und Fragmentierung von Wärme in der Trocknung werden die häufigsten Formen von Schäden, die Schaffung der so genannte 90 % DNA Überbrückungskupplung, die PCR-basierten Studien11belastet hat. Abgesehen von Fragmentierung ist das zweithäufigste Problem im Herbarium Genomics Verunreinigungen, wie z. B. die endophytische Pilze13 abgeleitet oder Pilze Obduktion erworben nach Sammlung aber vor dem Einbau in das Herbarium12, obwohl dieses Problem kann gelöst Bioinformatically rechts Pilze Datenbank (s.u.) gegeben werden. Eine dritte, und weniger üblich, Problem ist Sequenz Modifikation durch Cytosin Deamination (C/G→T/A)14, obwohl es wird geschätzt (~ 0,03 %) niedrig sein Herbarium Exemplare11. Mit dem Aufkommen von Hochdurchsatz-Sequenzierung (HTS) kann das Problem der Fragmentierung mit kurzen Lese- und Sequenzierung Tiefe12,15, so dass Genomebene Datenerfassung aus zahlreichen Proben mit niedriger Qualität überwunden werden DNA, und manchmal sogar ganze Genom-Sequenzierung15erlaubt.
Herbarium Proben werden immer häufiger verwendet und sind ein größerer Bestandteil der phylogenetische Projekte16. Eine aktuelle Herausforderung für HTS Herbar zu verwenden ist konsequent ausreichend Doppel stranded DNA, eine notwendige Voraussetzung für die Sequenzierung Protokolle, von zahlreichen Tierarten in einer fristgerechten Weise erhalten ohne Methoden zur individuellen Optimierung Proben. In diesem Papier wird ein Protokoll für die DNA-Extraktion und Bibliothek Vorbereitung der Herbarbelege demonstriert, nutzt die vorhandenen Methoden und ändert sie, um schnelle und reproduzierbare Ergebnisse zu ermöglichen. Diese Methode ermöglicht für die Komplettbearbeitung von Probe zu einer Bibliothek von 24 Proben in 13 h, mit Hands-on-Zeit 8 h oder 16 h, mit 9 h Arbeitsaufwand beträgt, wenn der optionale Reamplification-Schritt benötigt wird. Gleichzeitige Verarbeitung von mehr Proben ist erreichbar, wenn der limitierende Faktor Zentrifuge Kapazität und technisches Geschick. Das Protokoll soll nur typische Laborgeräte (Thermocycler, Zentrifuge und magnetische steht) anstelle von Spezialausrüstung, wie z. B. einen Vernebler oder Sonikator, für Scheren DNA benötigen.
DNA-Qualität, Fragmentgröße und Menge sind Faktoren für den Einsatz von Herbar im Hochdurchsatz-Sequenzierung Experimente begrenzt. Weitere Methoden zum Herbarium DNA zu isolieren und Erstellen von Hochdurchsatz-Sequenzierung Bibliotheken haben gezeigt, das Dienstprogramm zu verwenden so wenig wie 10 ng DNA-16; jedoch bedürfen experimentell ermitteln die optimale Anzahl von PCR-Zyklen für Bibliothek Vorbereitung erforderlich. Dies wird unpraktisch, beim Umgang mit äußerst geringen Mengen an lebensfähigen Doppel DNA (DsDNA), gestrandet, da einige Herbar nur genügend DNA für eine einzelne Bibliothek Zubereitung produzieren. Die hier vorgestellte Methode verwendet eine einzelne Zahl der Zyklen unabhängig von Sample-Qualität, so dass keine DNA Bibliothek Optimierungsschritte verloren geht. Stattdessen wird ein Reamplification Schritt aufgerufen, wenn Bibliotheken nicht die Mindestbeträge für die Sequenzierung erforderlich erfüllen. Viele Herbarium Proben sind selten und besitzen wenig Material macht es schwierig, destruktive Probenahme in vielen Fällen zu rechtfertigen. Um dem entgegenzuwirken, die vorgestellte Protokoll ermöglicht DsDNA Eingang Größen von weniger als 1,25 ng in der Bibliothek Vorbereitungsprozess, Erweiterung des lebensfähigen Proben für Hochdurchsatz-Sequenzierung und die Notwendigkeit einer destruktiven Entnahme von Proben zu minimieren.
Das folgende Protokoll wurde optimiert für Gräser und getestet auf Hunderte verschiedener Arten von Herbarium Proben, obwohl wir erwarten, dass das Protokoll auf viele andere Pflanzengruppen angewendet werden kann. Freuen Sie sich auf eine optionale Wiederherstellungsschritt, die verwendet werden, um niedrige Qualität und/oder seltene Exemplare zu speichern. Basierend auf mehr als zweihundert Herbar getestet, funktioniert dieses Protokoll auf Proben mit niedrigen Gewebe ein- und Qualität, so dass für die Erhaltung der seltene Exemplare durch minimale destruktive Probenahme. Hier wird gezeigt, dass dieses Protokoll hochwertige Bibliotheken bieten kann, die für Datenqualität-basierte Projekte sequenziert werden können.

<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

Herbarien sind eine unschätzbare Quelle von pflanzlichem Material, das in einer Vielzahl von biologischen Studien verwendet werden kann. Die Verwendung von Herbar ist verbunden mit einer Reihe von Herausforderungen einschließlich Probenqualität Erhaltung geschädigter DNA und destruktive Probenahme seltene Exemplare. Um effektiver Herbarium Material in großen Sequenzierung Projekten verwenden, ist eine zuverlässige und skalierbare Methode der DNA-Isolierung und Bibliothek Vorbereitung erforderlich. Dieses Papier zeigt eine robuste, Anfang-Ende Protokoll für DNA-Isolierung und hohem Durchsatz Bibliotheksbau von Herbar, die keine Änderung für Einzelproben erfordert. Dieses Protokoll ist für niedrige Qualität getrocknet Pflanze Material und nutzt vorhandene Methoden durch die Optimierung der Gewebe Schleifen, ändern Bibliothek Größenauswahl und die Einführung eines optionalen Reamplification Schritt für geringe Ausbeute Bibliotheken zugeschnitten. Reamplification von geringer Ausbeute DNA-Bibliotheken kann Proben abgeleitet von unersetzlichen und potenziell wertvollen Herbar, negiert die Notwendigkeit für zusätzliche destruktive Probenahme und ohne erkennbare Sequenzierung Voreingenommenheit für gemeinsame retten. phylogenetische Anwendungen. Das Protokoll ist auf Hunderten von Grasart geprüft worden, aber wird voraussichtlich nach Prüfung für den Einsatz in anderen pflanzlichen Linien angepasst werden. Dieses Protokoll kann durch extrem geschädigter DNA, wo Fragmente nicht in der gewünschten Größenordnung vorhanden sind, und sekundäre Pflanzenstoffe in einigen Pflanzenmaterial vorhanden, die saubere DNA-Isolierung hemmen begrenzt. Dieses Protokoll stellt insgesamt eine schnelle und umfassende Methode, die DNA-Isolierung und Bibliothek Vorbereitung von 24 Proben in weniger als 13 Stunden mit nur 8 h aktive Handhabungszeit mit minimalen Anpassungen ermöglicht.

DNA-Isolierung und endgültige Bibliothek Ertrag In dieser Studie wurde die Wirksamkeit des Protokolls für die Isolierung von Herbarium DNA und die Verwertung von hochwertigen Sequenzierung Bibliotheken nachgewiesen mit fünfzig verschiedenen Proben mit das älteste aus dem Jahre 1920 und der jüngste ab 2012 (Tabelle 2). Für jede Probe wurde etwa 10 mg Blattgewebe für DNA-Isolierung verwendet. Grüner Blattgewebe wurde bevorzugt, falls verfügbar, ...

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

Robuste DNA-Isolierung und Hochdurchsatz-Sequenzierung Bibliotheksbau für Herbar

Dieser Artikel beschreibt ein detailliertes Protokoll für DNA-Isolierung und Hochdurchsatz-Sequenzierung Bibliotheksbau aus Herbarium Material einschließlich Rettung von sehr schlechter Qualität 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 ...

robust-dna-isolation-high-throughput-sequencing-library-construction

Research

JoVE Journal

Genetics

10.5K Aufrufe.  Donald Danforth Plant Science Center. Dieser Artikel beschreibt ein detailliertes Protokoll für DNA-Isolierung und Hochdurchsatz-Sequenzierung Bibliotheksbau aus Herbarium Material einschließlich Rettung von sehr schlechter Qualität DNA.

Serie: 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.

Automatisierte Gel Größenauswahl, um die Qualität der nächsten Generation Sequencing Libraries von Umweltwasserproben vorbereitet verbessern

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

Forschung

Umwelt

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.

Reinigung von High Molecular Weight genomischer DNA aus Echten Mehltaus für Long-Read-Sequenzierung

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

Genetik

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.

Optimierung und Vergleichsanalyse von pflanzlichen organellaren DNA-Anreicherungsmethoden Geeignet für die Sequenzierung der nächsten Generation

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.

Empirische, metagenomischen und Computertechniken beleuchten die Mechanismen, durch welche Fungizide Kompromiss Bienengesundheit

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.

Frequenzverschiebungen Eingabe Genom Sequenzierung Bibliothek Vorbereitung von einem einzigen Tardigrade Exemplar

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.

Hybride De-Novo-Genomassemblierung zur Erzeugung vollständiger Genome von Harnbakterien unter Verwendung von Kurz- und Langzeitsequenzierungstechnologien

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 für Heilpflanzen: Identifizierung und phylogenetische Analyse

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>Autor im Rampenlicht: Nutzung der DNA-Barcode-Technologie zur Verbesserung der Effizienz der Identifizierung von Heilpflanzen

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.

Anwendung von DNA-Barcoding zur Identifizierung von Heilpflanzen

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: Eine Methode zur gleichzeitigen Sequenzierung Bibliothek Vorbereitung von DNA und RNA mit Hydrogel Matrizen

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 ...

Robuste DNA-Isolierung und Hochdurchsatz-Sequenzierung Bibliotheksbau für Herbar

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Automatisierte Gel Größenauswahl, um die Qualität der nächsten Generation Sequencing Libraries von Umweltwasserproben vorbereitet verbessern

Reinigung von High Molecular Weight genomischer DNA aus Echten Mehltaus für Long-Read-Sequenzierung

Optimierung und Vergleichsanalyse von pflanzlichen organellaren DNA-Anreicherungsmethoden Geeignet für die Sequenzierung der nächsten Generation

Empirische, metagenomischen und Computertechniken beleuchten die Mechanismen, durch welche Fungizide Kompromiss Bienengesundheit

Frequenzverschiebungen Eingabe Genom Sequenzierung Bibliothek Vorbereitung von einem einzigen Tardigrade Exemplar

Hybride De-Novo-Genomassemblierung zur Erzeugung vollständiger Genome von Harnbakterien unter Verwendung von Kurz- und Langzeitsequenzierungstechnologien

Anwendung von DNA-Barcoding zur Identifizierung von Heilpflanzen

Anwendung von DNA-Barcoding zur Identifizierung von Heilpflanzen

Anwendung von DNA-Barcoding zur Identifizierung von Heilpflanzen

Gel-Seq: Eine Methode zur gleichzeitigen Sequenzierung Bibliothek Vorbereitung von DNA und RNA mit Hydrogel Matrizen