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

Авторы благодарят за их техническую поддержку в области секвенирования геномных Nozomi Абэ, Юки Такаи и Уэхаси Исии. Эта работа была поддержана субсидий для японского общества для поощрения науки (JSP) научный сотрудник, KAKENHI субсидий для молодых ученых (No.22681029) и KAKENHI субсидий для научных исследований (B), № 17 H 03620 от JSP-страницы, по Грант для основных научно исследовательских проектов от Sumitomo фонда (No.140340) и частично путем исследования средств от правительства префектуры Ямагата и город Цуруока, Япония. Chlorella vulgaris используется для корма тихоходок была предоставлена любезно хлореллы Industry Co. LTD.

1. Подготовка
<ol><li>Готовить 2% агарозном геле с помощью дистиллированной воды (DW) как растворитель в 90-мм пластиковая культуры блюдо и 10 мл 1% пенициллина/стрептомицина коктейль с DW. Гель может храниться на 2-3 недели в инкубаторе на 18 ° C. Примечание: Избегайте любой хранения геля ниже 10 ° C,...

<ol>
	<li>Crowe, J. H., Hoekstra, F. A., Crowe, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis.">Anhydrobiosis.</a> Annual Review of Physiology. 54 (1), 579-599 (1992).</li><li>Mobjerg, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+in+extreme+environments+-+on+the+current+knowledge+of+adaptations+in+tardigrades.">Survival in extreme environments - on the current knowledge of adaptations in tardigrades.</a> Acta Physiologica. 202 (3), 409-420 (2011).</li><li>Becquerel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=La+suspension+de+la+vieau+dessous+de+1/20+K+absolu+par+demagnetization+adiabatique+de+L'alun+de+fer+dans+le+vide+les+plus+el&#233;ve.">La suspension de la vieau dessous de 1/20 K absolu par demagnetization adiabatique de L'alun de fer dans le vide les plus el&#233;ve.</a> Comptes Rendus de l'Acad&#233;mie des Sciences. 231, 261-264 (1950).</li><li>Ono, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ultra-high+pressure+on+small+animals,+tardigrades+and+Artemia.">Effect of ultra-high pressure on small animals, tardigrades and Artemia.</a> Cogent Physics. 3 (1), 1167575 (2016).</li><li>Horikawa, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+of+anhydrobiotic+eggs+of+the+Tardigrade+Ramazzottius+varieornatus+to+extreme+environments.">Tolerance of anhydrobiotic eggs of the Tardigrade Ramazzottius varieornatus to extreme environments.</a> Astrobiology. 12 (4), 283-289 (2012).</li><li>Horikawa, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+DNA+repair+and+protection+in+the+Tardigrade+Ramazzottius+varieornatus+and+Hypsibius+dujardini+after+exposure+to+UVC+radiation.">Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.</a> PLoS One. 8 (6), e64793 (2013).</li><li>Horikawa, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+tolerance+in+the+tardigrade+Milnesium+tardigradum.">Radiation tolerance in the tardigrade Milnesium tardigradum.</a> International Journal of Radiation Biology. 82 (12), 843-848 (2006).</li><li>May, R. M., Maria, M., Gumard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+diff&#233;rentielle+des+rayons+x+et+ultraviolets+sur+le+tardigrade+Macrobiotus+areolatus,+a+L'&#233;tat+actif+et+dess&#233;ch&#233;.">Action diff&#233;rentielle des rayons x et ultraviolets sur le tardigrade Macrobiotus areolatus, a L'&#233;tat actif et dess&#233;ch&#233;.</a> Bulletin Biologique de la France et de la Belgique. 98, 349-367 (1964).</li><li>Jonsson, K. I., Harms-Ringdahl, M., Torudd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+tolerance+in+the+eutardigrade+Richtersius+coronifer.">Radiation tolerance in the eutardigrade Richtersius coronifer.</a> International Journal of Radiation Biology. 81 (9), 649-656 (2005).</li><li>Bemm, F., Weiss, C. L., Schultz, J., Forster, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+of+a+tardigrade:+Horizontal+gene+transfer+or+bacterial+contamination?.">Genome of a tardigrade: Horizontal gene transfer or bacterial contamination?.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (22), E3054-E3056 (2016).</li><li>Delmont, T. O., Eren, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+contamination+with+advanced+visualization+and+analysis+practices:+metagenomic+approaches+for+eukaryotic+genome+assemblies.">Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies.</a> PeerJ. 4, e1839 (2016).</li><li>Koutsovoulos, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+extensive+horizontal+gene+transfer+in+the+genome+of+the+tardigrade+Hypsibius+dujardini.">No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (18), 5053-5058 (2016).</li><li>Boothby, T. C., Goldstein, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reply+to+Bemm+et+al.+and+Arakawa:+Identifying+foreign+genes+in+independent+Hypsibius+dujardini+genome+assemblies.">Reply to Bemm et al. and Arakawa: Identifying foreign genes in independent Hypsibius dujardini genome assemblies.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (22), E3058-E3061 (2016).</li><li>Boothby, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+extensive+horizontal+gene+transfer+from+the+draft+genome+of+a+tardigrade.">Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (52), 15976-15981 (2015).</li><li>Arakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+extensive+horizontal+gene+transfer+from+the+draft+genome+of+a+tardigrade.">No evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (22), E3057 (2016).</li><li>Arakawa, K., Yoshida, Y., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequencing+of+a+single+tardigrade+Hypsibius+dujardini+individual.">Genome sequencing of a single tardigrade Hypsibius dujardini individual.</a> Scientific Data. 3, 160063 (2016).</li><li>Yoshida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+of+the+tardigrades+Hypsibius+dujardini+and+Ramazzottius+varieornatus.">Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.</a> PLoS Biology. 15 (7), e2002266 (2017).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+RNA+Extraction+from+C.+elegans.">Total RNA Extraction from C. elegans.</a> Bio-protocol. Bio101, e47 (2011).</li><li>Andrews, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FastQC a quality-control tool for high-throughput sequence data. , (2015).</li><li>Hashimoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremotolerant+tardigrade+genome+and+improved+radiotolerance+of+human+cultured+cells+by+tardigrade-unique+protein.">Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.</a> Nature Communications. 7, 12808 (2016).</li><li>Zimin, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MaSuRCA+genome+assembler.">The MaSuRCA genome assembler.</a> Bioinformatics. 29 (21), 2669-2677 (2013).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25 (14), 1754-1760 (2009).</li><li>Okonechnikov, K., Conesa, A., Garcia-Alcalde, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualimap+2:+advanced+multi-sample+quality+control+for+high-throughput+sequencing+data.">Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data.</a> Bioinformatics. 32 (2), 292-294 (2016).</li><li>Horikawa, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+rearing+system+of+the+extremotolerant+tardigrade+Ramazzottius+varieornatus:+a+new+model+animal+for+astrobiology.">Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: a new model animal for astrobiology.</a> Astrobiology. 8 (3), 549-556 (2008).</li></ol>

Бактериальное загрязнение представляет собой угрозу для геномные последовательности микроскопических организмов. В то время как предыдущие исследования на tardigrade генома отфильтрованы загрязнения, используя обширные информатики методы12,20, мы последов?...

Тихоходки являются микроскопических животных, которые могут вводить состояние ametabolic, называется anhydrobiosis, когда сталкиваются усыхания. Они восстановить путем поглощения воды1,2. В ametabolic состоянии тихоходки способны терпеть различных экстремальных средах, которые включают экстремальных температур3 и давления4,5, высокие дозы ультрафиолетового света6, рентгеновские и гамма-лучи 7 , 8и9космического пространства. Геномных данных является необходимой основой для изучения молекулярных механизмов anhydrobiosis.
Предыдущие попытки последовательности генома тихоходок показали признаки бактериального загрязнения10,11,12,,1314. Геномные последовательности от таких малых организмов требуется большое количество животных и подвержен бактериального загрязнения; Таким образом мы создали ранее последовательности протокол, с помощью сверхнизких метода ввода, начиная от единичная тихоходки, чтобы свести к минимуму риск загрязнений15. Используя эти данные, мы также провели ампликонов высокого качества и сборка генома H. dujardini16,17. Здесь мы подробно описать этот метод геномной последовательности от отдельного tardigrade ()рис. 1). Проверка этого метода секвенирования находится за пределами фокус этой бумаги и уже тщательно обсуждались в наших предыдущих доклада16.
Этот метод состоит из двух частей: изоляции одного тихоходки с самые низкие возможные загрязнения и извлечение высокое качество пиктограммы уровней ДНК. Тихоходки голодали и тщательно промыть водой, а также антибиотики и наблюдается под микроскопом с 500 кратном обеспечить устранение любых бактериального загрязнения. Предыдущие оценки и измерения показывают, что одного человека тихоходки содержит приблизительно 50-200 pg геномной ДНК16, который добывается путем крекинга хитин экзоскелет циклов замораживания оттаивания или ручной гомогенизации. Этот геномной ДНК представлен библиотеки строительных и виртуализации на инструменте секвенирования ДНК. Дополнительные информатики анализ показывает последовательность высокого качества, а также низкий уровень загрязнения по сравнению с предыдущими проектами tardigrade последовательности.

<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

Тихоходки являются микроскопических животных, которые переходят в состояние ametabolic, называется anhydrobiosis, когда сталкиваются усыхание и может вернуться к их первоначальное состояние, когда вода поставляется. Геномные последовательности микроскопических животных, таких как тихоходок риски бактериального загрязнения, что иногда приводит к ошибочной интерпретации, например, о масштабах горизонтальный перенос генов в этих животных. Здесь мы предоставляем сверхнизких метод ввода последовательности генома тихоходки, Hypsibius dujardini, от одного образца. Применяя строгие мытья и загрязнения изоляции наряду с эффективной добычи 50 ~ 200 pg геномной ДНК от одного лица, мы построили библиотеку виртуализации с помощью инструмента секвенирования ДНК. Эти библиотеки были весьма воспроизводимые и объективной, и информатики анализ последовательного читает с других геномов H. dujardini показал минимальное количество загрязнения. Этот метод может быть применен к unculturable тихоходок, которые не могут быть виртуализации с помощью предыдущих методов.

Изоляция загрязняющих веществ:
Этот протокол включает в себя тщательное мытье тихоходки и стерилизации с лечения антибиотиками для сведения к минимуму загрязнения. Она также включает в себя визуальный процесс проверки для о?...

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

Сверхнизкое ввода геном последовательность подготовки библиотеки от Tardigrade единичная

Большой проблемой остается загрязнение в ходе секвенирования геномных микроскопических организмов. Здесь мы покажем способ последовательности генома тихоходки от одного образца с всего лишь 50 pg геномной ДНК без усиления весь геном свести к минимуму риск загрязнения.

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

Обнаружение Copy Number Изменений Использование Single Cell Секвенирование

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

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Сопоставление взаимодействия РНК-РНК в глобальном масштабе с использованием биотинилированного псоралена

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

Поколения родной Chromatin иммунопреципитации секвенирования библиотек для анализа плотности нуклеосома

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

Универсальный протокол для крупномасштабных gRNA библиотека производства из любого источника ДНК

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: Высокая пропускная способность на основе последовательности техника для выявления поздно репликации областей генома

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

Надежные ДНК изоляции и высокопроизводительного секвенирования строительство библиотеки для образцов гербария

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

Ретроспективное микроРНК последовательности: Комплементарной ДНК библиотеки подготовка протокол с помощью образцов формалин Исправлена парафин врезанных РНК

&#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

Обнаружение редких событий, с помощью Исправлена ошибка ДНК и РНК последовательности

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

Секвенирование мРНК из цельной крови с использованием секвенирования Наноруды

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

Одноклеточная РНК секвенирование и анализ островков поджелудочной железы человека

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