破壊的なサンプリングの標本へのアクセスのためのミズーリの植物園とサンプリング標本、援助のクリスティーナ Zudock ヨルダン Teisher、テイラー AuBuchon-高齢者を感謝いたします。この作品は全米科学財団 (DEB-1457748) からの助成金による支援です。

<ol>
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著者は、彼らが競合する利益あるを宣言します。

ここで提示されたプロトコルは、DNA の隔離および植物の乾燥標本からのライブラリの準備をシーケンス処理の包括的かつ堅牢な方法です。メソッドとそれを変更する必要は最小限の一貫性は、それは大規模な標本に基づくシーケンス プロジェクトのスケーラブルな標本の品質確認に基づいています。低降伏点ライブラリのオプション reamplification ステップを含めることは、低品質、低量、珍...

標本コレクションは、種と系統1,2,3, 集団遺伝学4,5, 保全を含む研究のためのゲノムの多様性の両方の可能性がある貴重な情報源生物学6、外来種生物学7、および形質進化8。種、人口、地理的な場所、タイム ポイントの豊かな多様性を取得する機能は、標本は、「宝箱」9を強調表示します。歴史的に、標本由来 DNA の劣化した自然が多い領域の葉緑体ゲノムや、リボソームの内部転写スペーサー (ITS) などの高いコピー内で見つかったマーカーのみを使用する研究者を追いやって、PCR ベースのプロジェクトを妨げています。RNA。標本と DNA の品質によって異なります広く保全9,10二本鎖切断と損傷の最も一般的な形であること、作成の乾燥工程で使用される熱から断片化の手法、いわゆる 90 %dna ロックアップ PCR ベースの研究11を妨害しています。断片化、別標本のゲノムの第 2 最も一般的な問題は汚染、生菌13から派生する菌類コレクション後、標本12にもマウントする前に死後を取得したようにこの問題は、右真菌データベース (下記参照) を与えられた解決の bioinformatically をすることができます。11押し葉標本の低い (〜 0.03%) と推定されている、3 番目、およびより少なく共通の問題はシトシン脱アミノ化 (C/G→T/A)14日シーケンス変更です。短いリードとシーケンス深さ12,15、低品質の多数の標本から遺伝子レベルのデータ集録が可能高スループット シーケンス (HTS) の出現により、断片化の問題を克服できます。DNA と時々 全ゲノム シーケンス15を許可します。
標本サンプルはより頻繁に使用されるなる、系統のプロジェクト16の大きなコンポーネントです。標本を用いた高温超伝導体の現在の課題が個々 のための方法を最適化することがなく、タイムリーに多くの種から十分な二本鎖 DNA、シーケンス プロトコルは、必要な前提条件を一貫して取得します。標本。本稿では、既存の手段を活用して、高速かつ複製可能な結果を可能にするそれらを変更に DNA の抽出と標本のライブラリの準備のためのプロトコルが示されています。この方法は 13 h、8 h のハンズオン タイム、または 16 h、9 h、ハンズオン時間、オプション reamplification ステップが必要な場合 24 サンプルのライブラリに試験片から処理が完了すること。多くのサンプルの同時処理は、制限要因は遠心力と技術的なスキルが達成可能で。プロトコルは DNA をせん断してだけ典型的な実験装置 (たち、遠心とマグネット スタンド) ネブライザー、超音波発生装置などの特殊な機器ではなくを必要とする設計されています。
DNA の品質、フラグメント サイズと数量は、高スループット シーケンス実験の標本の使用のための要因を制限しています。標本の DNA を分離し、高スループット シーケンス ライブラリを作成する他の方法は 10 として少し使用の有用性を実証している DNA16; ngただし実験的 PCR の最適な数を決定する必要なライブラリの準備のために必要なサイクルです。これは、いくつか標本が単一のライブラリの準備のため十分な DNA のみを生成するよう実行可能なダブルの非常に小さな金額を扱う鎖 DNA (dsDNA)、非現実的ななります。ライブラリの最適化の手順で DNA は失われませんので、ここで紹介した方法は単一サンプルの質に関係なくサイクル数を使用します。代わりに、ライブラリは、シーケンスに必要な最低限の量を満たしていない場合に、reamplification ステップが呼び出されます。多くの植物標本サンプルはまれであり、多くの場合有害なサンプリングを正当化するが難しく少し材料を所有しています。これを対抗するため提案するプロトコルにより、dsDNA 入力サイズを 1.25 未満 ng のライブラリの準備プロセス、高スループット シーケンスと供試体の破壊のサンプリングのための必要性を最小限に抑えるのための実行可能なサンプルの範囲を拡大します。
次のプロトコルを草用に最適化されており、我々 はプロトコルことができます他の多くの植物のグループに適用されることを期待するものの、標本サンプルから別の種の何百ものテストします。低品質または珍しい標本を保存するために使用できる省略可能な回復手順が含まれています。テスト 200 以上の標本を基に、このプロトコルは、最小限の破壊的なサンプリングによって珍しい標本の保存を可能にする低組織入力と品質、標本で動作します。ここでこのプロトコルがゲノミクス ベース プロジェクトのシーケンスことができます高品質ライブラリを提供できることを示す.

<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

標本は、さまざまな生物学的研究で使用できる植物材料の非常に貴重な源です。標本の使用はサンプル保存品質、劣化 DNA、珍しい標本の破壊的なサンプリングを含む課題の番号に関連付けられます。効果的に大規模なプロジェクトの標本材料を使用、するために DNA の分離およびライブラリの準備の信頼性と拡張性の高い方法が必要です。本稿は、DNA 分離、高速ライブラリ構築に標本から個々 のサンプルの変更を必要としない堅牢で、始めから終わりまでプロトコルを示します。このプロトコルは、組織研削、ライブラリ サイズ選択を変更して低収量ライブラリのオプション reamplification ステップを導入することを最適化することにより従来の低品質の乾燥植物素材とを利用に合わせて調整します。低収量 DNA ライブラリの reamplification は、かけがえのない、潜在的に貴重な標本、追加破壊サンプリングし、共通認識できるシーケンス バイアスを導入することがなく、必要性を否定することから派生したサンプルを救うことができます。系統のアプリケーション。プロトコル数百草種でテストされていますが確認後他の植物の系統での使用に適応します。このプロトコルは、フラグメントは目的のサイズの範囲では存在しない、非常に低下した DNA ときれいな DNA 分離を抑制する二次代謝産物いくつかの植物材料の存在によって制限することができます。全体的に、このプロトコルは DNA の隔離と最小限の変更でアクティブなハンズオン タイムの 8 時間だけで 13 h 未満の 24 サンプル調製されたライブラリでは、高速かつ包括的な方法を紹介します。

DNA の隔離および最終的なライブラリ収量 本研究では 1920 年からの最も古いと 2012 年 (表 2) から最年少 50 の異なるサンプルを使用して標本の DNA の分離および高品質シーケンス ライブラリの回復用のプロトコルの有効性を示した.各サンプルは、葉の組織の約 10 mg は DNA の隔離に使われました。場合は、環境に優しい葉組織が支持され、明?...

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

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

この記事では、DNA の隔離及び非常に質の悪い DNA の救助を含む標本材料から高スループット シーケンス ライブラリ構築のための詳しいプロトコルを示します。

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 ビュー.  Donald Danforth Plant Science Center. この記事では、DNA の隔離及び非常に質の悪い DNA の救助を含む標本材料から高スループット シーケンス ライブラリ構築のための詳しいプロトコルを示します。

ビデオ: 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.

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

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

環境学

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.

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

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

遺伝学

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.

次世代シークエンシングに適した植物オルガネラDNA濃縮法の最適化と比較分析

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.

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

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.

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

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.

短読シーケンシング技術を用いた尿細菌の完全ゲノム生成のためのハイブリッド デノボ ゲノムアセンブリ

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>薬用植物のDNAバーコード化:同定と系統解析

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>著者スポットライト:DNAバーコード技術を活用して薬用植物同定の効率を高める

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.

薬用植物同定のためのDNAバーコードの応用

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.

ゲル-seq: DNA および RNA のゲルのマトリックスを使用しての同時配列ライブラリの準備の方法

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 ...

堅牢な DNA の隔離および標本の高スループットシーケンスライブラリーの構築

要約

さらに動画を探す

この動画の章

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

次世代シークエンシングに適した植物オルガネラDNA濃縮法の最適化と比較分析

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

短読シーケンシング技術を用いた尿細菌の完全ゲノム生成のためのハイブリッドデノボゲノムアセンブリ

薬用植物同定のためのDNAバーコードの応用

薬用植物同定のためのDNAバーコードの応用

薬用植物同定のためのDNAバーコードの応用

ゲル-seq: DNA および RNA のゲルのマトリックスを使用しての同時配列ライブラリの準備の方法

堅牢な DNA の隔離および標本の高スループット シーケンス ライブラリーの構築

要約

さらに動画を探す

この動画の章

環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択

ロングリード配列決定のためのうどんこ病から高分子量のゲノムDNAの精製

次世代シークエンシングに適した植物オルガネラDNA濃縮法の最適化と比較分析

実証、メタゲノムと計算手法を照らす殺菌剤妥協ミツバチ健康メカニズム

超低入力ヒトゲノム単一 Tardigrade の標本からのライブラリの準備

短読シーケンシング技術を用いた尿細菌の完全ゲノム生成のためのハイブリッド デノボ ゲノムアセンブリ

薬用植物同定のためのDNAバーコードの応用

薬用植物同定のためのDNAバーコードの応用

薬用植物同定のためのDNAバーコードの応用

ゲル-seq: DNA および RNA のゲルのマトリックスを使用しての同時配列ライブラリの準備の方法

堅牢な DNA の隔離および標本の高スループットシーケンスライブラリーの構築

短読シーケンシング技術を用いた尿細菌の完全ゲノム生成のためのハイブリッドデノボゲノムアセンブリ