Name: ultralow input genome sequencing library preparation from a single tardigrade specimen
Uploaded: 2018-07-15T14:00:00
Description: Watch this Scientific Journal Video about Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen at JoVE.com

著者はゲノム配列決定でテクニカル サポートのため希阿部、希高井菜穂子石井をありがちましょう。この作業によって支えられた費日本社会のための科学振興会研究員、科研費科研費若手 (No.22681029)、科研費科研費研究 (B)、第 17 H 03620、日本学術振興会からので、基本的な科学研究プロジェクト、住友財団 (No.140340) から、山形県・鶴岡市から研究資金によって部分的に助成。クロレラクマムシをフィードするために使用されたベルフォートは、クロレラ工業 (株)

1. 準備
<ol><li>90 mm プラスチック製培養皿で 10 mL、1% ペニシリン/ストレプトマイシン DW のとカクテルの溶媒として蒸留水 (DW) を使用して 2% の agarose のゲルを準備します。ゲルは 18 ° C で設定インキュベーターで 2 〜 3 週間に保存できます。 注: は、低温は agarose のゲル、ゲルと、クマムシをトラップできる培養皿の壁の非常に小さいギャップの結果を縮小するための 10 の ° C の下のゲル?...

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細菌汚染は、微生物のゲノム配列決定に脅威を与えます。Tardigrade ゲノムに関する先行研究を広範な情報学研究の方法12,20を使用して汚染をフィルター処理、一方我々 は汚染のリスクを最小限に抑えるために単一の個人からゲノムをシーケンスです。個々 緩歩動物ゲノム DNA16の約 50-200 pg が含まれているキチン質の外骨格の厚い層...

クマムシは、乾燥に直面するとき、乾燥耐性と呼ばれる ametabolic 状態を入力することができます顕微鏡動物です。彼らは水1,2の吸収による回復します。Ametabolic 状態ではクマムシが極端な温度3高圧4、5ガンマ線、x 線、紫外線光6の高用量を含む様々 な極限環境に耐えることができます。7,8、および宇宙空間9。ゲノムデータは乾燥耐性の分子機構の研究のために不可欠な基礎です。
クマムシのゲノムをシーケンス処理する前の試みは、細菌汚染10、11,12,13,14の兆候を示しています。このような小さな生物からゲノム配列決定多数の動物を必要とし、細菌汚染になりやすいです。したがって、我々 は既に15汚染のリスクを最小限に抑えるため、クマムシの単一の標本から超低入力メソッドを使用してシーケンス プロトコルを確立されています。これらのデータを使用して、さらに実施した高品質の順序とH. dujardini16,17のゲノムの再構築。ここで述べるこの(図 1tardigrade 一個人からゲノム配列決定法)。このシーケンスの結果の検証は本稿の焦点を越えて、私たちの以前のレポート16で既に、徹底的に議論されています。
このメソッドは、2 つの部分で構成されています: 最低汚染可能と DNA のピクトグラム レベルの質の高い抽出単一クマムシの分離。海中でもは飢えたと抗生物質と同様に、水ですすぎ、任意の細菌汚染の除去を確実に 500 倍の倍率での顕微鏡で観察しました。以前の推定値と測定、クマムシの単一の個人にゲノム DNA の16、凍結融解のサイクルによってまたは手動の均質化によって、キチン質の外骨格を割れによって抽出される約 50 200 pg が含まれていることを示しています。この DNA は、図書館の建設に提出し、DNA シーケンシング装置でシーケンス処理します。追加情報分析は、以前の tardigrade シーケンス プロジェクトと比較して汚染の低レベルと同様、高品質のシーケンスを示しています。

<table border="0" cellpadding="0" cellspacing="0" style="width:895px;" width="894">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">SZ61 microscope</td>
			<td style="width:251px;">OLYMPUS</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BactoAgar</td>
			<td style="width:251px;">Difco Laboratories</td>
			<td align="right" style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Penicillin Streptomycin (10,000 U/mL)</td>
			<td style="width:251px;">Gibco by life technologies</td>
			<td style="width:119px;">15140-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">VHX-5000 System</td>
			<td style="width:251px;">Keyence</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">0.2mL Silicone coating tube</td>
			<td style="width:251px;">Bio Medical Science</td>
			<td style="width:119px;">BC-bmb20200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Quick-DNA Microprep Kit</td>
			<td style="width:251px;">ZYMO Research</td>
			<td style="width:119px;">D3021</td>
			<td>Use of this kit is absolutey critical; see step 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">1.5 mL microtube</td>
			<td style="width:251px;">greiner bio-one</td>
			<td style="width:119px;">616-201</td>
			<td>See 4.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HIgh speed refrigerated micro centrifuge</td>
			<td style="width:251px;">TOMY</td>
			<td style="width:119px;">MX-307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Covaris M220</td>
			<td style="width:251px;">Covaris Inc.</td>
			<td align="right" style="width:119px;">4482277</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">ThruPLEX DNA-Seq kit</td>
			<td style="width:251px;">Rubicon Genomics</td>
			<td style="width:119px;">CAT. NO. R400406</td>
			<td>Use of this kit is absolutey critical; see step 4.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thermal Cycler</td>
			<td style="width:251px;">Bioer Technology</td>
			<td style="width:119px;">TC-96GHbC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">AMPure XP reagent</td>
			<td style="width:251px;">BECKMAN COULTER Life Science</td>
			<td style="width:119px;">A63881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ethanol</td>
			<td style="width:251px;">Wako</td>
			<td style="width:119px;">054-027335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">EB buffer</td>
			<td style="width:251px;">QIAGEN</td>
			<td align="right" style="width:119px;">19086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">2200 TapeStation</td>
			<td style="width:251px;">Agilent</td>
			<td style="width:119px;">G2965AA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D1000 Reagents</td>
			<td style="width:251px;">Agilent</td>
			<td style="width:119px;">5067-5583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D1000 ScreenTape</td>
			<td style="width:251px;">Agilent</td>
			<td style="width:119px;">5067-5582</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Qubit dsDNA BR Buffer/Reagent</td>
			<td style="width:251px;">ThermoFisher Scientific</td>
			<td style="width:119px;">Q32850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cubee Mini-Centrifuge</td>
			<td style="width:251px;">RecenttecGenereach</td>
			<td style="width:119px;">R5-AQBD01aqbd</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MiSeq 600 cycle v3</td>
			<td style="width:251px;">Illumina Inc.</td>
			<td style="width:119px;">MS-102-3003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">MiSeq Sequencer</td>
			<td style="width:251px;">Illumina Inc.</td>
			<td style="width:119px;">SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultralow input genome sequencing library preparation from a single tardigrade specimen

クマムシは、乾燥とすることができますに直面するときと呼ばれる乾燥耐性を元の状態に返すときに水が供給されて ametabolic 状態顕微鏡動物です。時々 これらの動物の遺伝子の水平伝達の範囲についてたとえば、誤った解釈につながるクマムシ リスク細菌汚染など顕微鏡動物のゲノム配列を決定。ここでは、我々 は単一の標本からHypsibius dujardiniクマムシのゲノムを配列する超低入力メソッドを提供します。50 の効率的な抽出と厳密な洗浄および汚染物質の除外を用いて 〜 200 pg 単一の個人からのゲノム DNA、DNA シーケンシング装置でシーケンス化されたライブラリを構築しました。これらのライブラリは、高い再現性で、公平をされ他h. dujardiniのゲノム シーケンスされた読み取りのインフォマティクス解析は汚染の最小限の量を示した。このメソッドは、以前の方法を使用していないシーケンスが難のクマムシに適用できます。

汚染物質の除外:
このプロトコルには、海中でも、汚染を最小限に抑えるために抗生物質治療と殺菌の徹底した洗浄が含まれます。また、これらのプロセスの完全性を確保するため、視覚的なチェック プロセスが含まれます。検証 (ステップ 2.4 プロトコルの) 中に作られた顕微鏡イメージは図 ...

Watch this Scientific Journal Video about Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen at JoVE.com

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

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

顕微鏡の有機体のゲノムのシーケンス処理中に汚染大きい問題に残る。ここでは、汚染のリスクを最小限に抑えるため全ゲノム増幅 DNA のわずか 50 pg を単一の標本からクマムシのゲノムをシーケンス処理する方法を示します。

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

The goal of this protocol is to sequence the genome of a microscopic organism called tardigrades. We have established a method to sequence the genome of a tardigrade, Hypsibius dujardini, from a single specimen with as low as 50 picograms of genomic DNA with a whole genome application. After isolating a single tardigrade, we minimize bacterial contamination by using antibiotics and visual inspection.We have also utilized two homogenization methods. First and most commonly used in C.elegans using freeze and thaw cycles, and second, a manual crushing of the tardigrade with a pipette tip. DNA is then used to construct a sequencing library and then is sequenced in a MiSeq Instrument.An overall summary of the protocol. After isolation of a single individual, it is subjected to three freeze and thaw cycles for homogenization. The genomic DNA is extracted and purified and is fragmented by sonication.Then a sequencing library is constructed, and after validation of library size distribution, it is sequenced with a sequencing instrument. Prepare 2%agarose gel using distilled water as the solvent in a 90 millimeter plastic culture dish, and 10 millimeters of 1%penicillin streptomycin with distilled water. The gel can be stored for two to three weeks in an incubator set at 18 degrees.Collect a single tardigrade and place on the prepared agar plate, and wash with distilled water two to three times to remove remaining particles. Place the single tardigrade in penicillin streptomycin antibiotics for two to six hours to remove bacterial contamination, and place the decontaminated animal on a clean slide glass using a P10 pipette. Observe the tardigrade under a microscope at 500 times magnification, and confirm that there are no remaining bacteria.Collect individual using a P10 pipette with maximum of five microliters of liquid, and place into a low-binding PCR tube, and remove excess liquid as much as possible. Homogenization and DNA extraction. Homogenize the animal to obtain genomic DNA with one of the following methods.Homogenization with freeze and thaw cycles. Immediately following step 2.5, add 100 microliters of lysis buffer to the PCR tube containing the tardigrade. Place the PCR tube in liquid nitrogen for 10 minutes, and move to a heat block, warm to 37 degrees for 10 minutes.Repeat this step three times. Manual crushing. Under a stereo microscope, crush the individual with a P10 pipette tip by pressing the animal against the PCR tube wall, and immediately add 100 microliters of lysis buffer.Incubate for 30 minutes at room temperature for lysis to occur. Transfer the complete volume of the lysis mixture to a clean 1.5 milliliters low-binding microtube. Add 100 microliters of lysis buffer to the low-binding PCR tube, which is used for homogenization and is now empty.And after pipetting, transfer the mixture to 1.5 low-binding microtube. Repeat this step twice. Add 300 microliters of lysis buffer to a low-binding PCR tube, and after pipetting, move the mixture to 1.5 milliliter low-binding microtube.Add the total of 600 microliters lysis mixture to spin column placed in the collection tube, and centrifuge at 10, 000 G for one minute. Reapply the flow through to the column, and centrifuge at 10, 000 G for one minute. This step is critical to ensure that most of the genomic DNA is bound to the column.Add 500 microliters of wash buffer to the spin column, and centrifuge at 10, 000 G for one minute. Transfer the spin column to a clean 1.5 milliliter microtube. Apply 20 microliters of 10 millimolar first ACL to the spin column and wait for five minutes at room temperature.Centrifuge at 10, 000 G for one minute. The dilution buffer must not contain EDTA, for it interferes with laboratory preparation enzymes. Reapply the flow through to the spin column, and after five minutes incubation at room temperature, centrifuge for one minute at 10, 000 G.Sequencing library construction.DNA fragmentation. Transfer 15 microliters of genomic DNA elute To a 15 microliter microtube for DNA fragmentation, and centrifuge for one minute using a tabletop centrifuge. Fragment the genomic DNA to 550 base pairs.After through pipetting, transfer 10 microliters of fragmented DNA mixture to a clean, low-binding PCR tube. Experiment can be stopped here. Preserve DNA at four or minus 20 degrees.Sequencing library construction. It is absolutely critical to use the specified kit in the following procedures, due to low-input DNA. Prepare the reagents required, following the manufacturer's protocol, and construct the sequencing library without any modifications.After applying library amplification mixture to a library synthesis mixture, perform PCR reaction in a thermal cycler. The PCR reaction was conducted as shown. Purification of the PCR reaction.Add 50 microliters of magnetic beads and pipette 10 times, and centrifuge briefly with a tabletop micro centrifuge. Incubate for two minutes at room temperature. Incubate on magnetic stand for five minutes, or until the solution becomes completely clear, and remove the supernatant.Follow the manufacturer's protocol. Transfer the supernatant without disturbing the pellet into a new low-binding PCR tube. DNA quality check and quantification, and sequencing.Validation of DNA library size distribution. Add three microliters of sample water with one microliter of sequencing library, and mix thoroughly for one minute with a vortex, and briefly centrifuge with a tabletop centrifuge. Conduct electrophoresis and validate the library size distribution with associated software.The main fragment peak should be broadly ranging from about 300 to 1, 000 base pairs. DNA quantification. Add 796 microliters of the solution buffer and four microliters of the fluorescence reagent and mix thoroughly.Dispense 190 microliters of the working solution to two assay tubes, and 197 microliters to one assay tube. Add 10 microliters of standards with known concentrations of DNA to each assay tube containing 190 microliters of working solution, and three microliters of the prepared library to assay tube containing 197 microliters of working solution. Vortex briefly, and centrifuge on the tabletop centrifuge.Quantify the DNA using a fluorometer with three microliter settings. Sequencing of the DNA library. Prepare the sequencing library based on the manufacturer's protocol.Set the reagent cassette and flow cell into the sequencing instrument, and enter the sequencing running information, following the manufacturer's protocol. Run sequencing. Represented results.Exposure of contaminants in high-quality DNA extraction remains to be the critical point in our protocol. To visually examine if they're contaminant, we have incubated the tardigrade in antibiotics, which are penicillin and streptomycin, and also examined the individual under a 500 times microscope. As shown here, visible microbes around the tardigrades are completely illuminated.Following efficient homogenization, DNA extraction, fragmentation, and library preparation. The size distribution of the library is validated using a high sensitive electrophoresis for quality control. The purple and green lines indicate upper and lower markers at 1, 500 and 25 base pairs respectively.Lane L is a ladder marker, lane S and N1 to N4 are five replicates of the same experiment, all starting from a single tardigrade specimen. As it can be seen from the broad uniform and distribution between 200 and 1, 000 base pairs, our protocol is highly reproducible, even with the ultra-low input. Here's the result of fast QC, for synchronized reads obtained from a single sequencing run.Reads are obtained as spare ends of 300 base pairs, where forward and reverse reads are shown in top and bottom, respectively. The distribution shown here is typical of 300 pair end reads, with very high quality base calling above Q30 for first 200 base pairs, and gradually declining to the end. So this method uses only a single individual for one sample.There is no requirements of massive amounts of animals, such as those required in previous tardigrade genome sequencing projects. Culturing methods for most tardigrades have not yet been established. Therefore, enabling genomics sequencing from a single individual, such as those directly from the fieldwork, will have a large impact on tardigrade molecular biology, and possibly for other small animals.Our DNA sequencing methods makes it possible to analyze other numerous microscopic organisms that have not yet been sequenced, such as rare hogweed species, nematodes, water firs, and other options, by connecting the part of the zones and a wider public area, furthering the setting where various biological mechanisms may be possible.

Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

単一細胞のシーケンシングを使用したコピー数変化の検出

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

遺伝学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

ビオチン化ソラレンを用いたRNA-RNA相互作用のマッピング

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

ネイティブのクロマチン免疫沈降のヌクレオソーム密度解析ライブラリをシーケンスの生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

大規模な gRNA ライブラリ生産 DNA の任意のソースからの普遍的なプロトコル

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 高スループット シーケンス ベースを識別する方法後半ゲノムの領域を複製します。

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

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

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 ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

エラー修正 DNA ・ RNA シーケンスを用いた珍しいイベント検出

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

ナノポアシーケンシングを用いた全血からの mRNA の配列決定

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

ヒト膵島の単一細胞RNAシーケンシングと解析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...