The authors thank Nozomi Abe, Yuki Takai, and Nahoko Ishii for their technical support in genomic sequencing. This work was supported by Grant-in-Aid for the Japan Society for the Promotion of Science (JSPS) Research Fellow, KAKENHI Grant-in-Aid for Young Scientists (No.22681029), and KAKENHI Grant-in-Aid for Scientific Research (B), No. 17H03620 from the JSPS, by a Grant for Basic Science Research Projects from The Sumitomo Foundation (No.140340), and partly by research funds from the Yamagata Prefectural Government and Tsuruoka City, Japan. Chlorella vulgaris used to feed the tardigrades was provided courtesy of Chlorella Industry Co. LTD.

1. Preparation
<ol>
	<li>Prepare 2% agarose gel using distilled water (DW) as the solvent in a 90-mm plastic culture dish, and 10 mL of a 1% penicillin/streptomycin cocktail with DW. The gel can be stored for 2 - 3 weeks in an incubator set at 18 &#176;C. 
	NOTE: Avoid any storage of the gel below 10 &#176;C, for the low temperature will shrink the agarose gel, resulting in a minuscule gap between the gel and the culture dish wall in which the tardigrades can be trapped.</li>
</ol>
2. Sample ...

<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>

The authors have nothing to disclose.

Bacterial contamination poses a threat to the genomic sequencing of microscopic organisms. While previous studies on tardigrade genome sequencing have filtered out contamination using extensive informatics methods12,20, we sequenced the genome from a single individual to minimize the risk of contaminations. Since an individual tardigrade contains approximately 50 - 200 pg of genomic DNA16 and is encased in a thick layer of chitin exoskelet...

Tardigrades are microscopic animals that can enter an ametabolic state called anhydrobiosis when facing desiccation. They recover by the absorption of water1,2. In the ametabolic state, tardigrades are capable of tolerating various extreme environments, which include extreme temperatures3 and pressures4,5, a high dosage of ultraviolet light6, X-rays, and gamma rays7,8, and cosmic space9. Genomic data is an indispensable foundation for the study of molecular mechanisms of anhydrobiosis.
Previous attempts to sequence the genome of tardigrades have shown signs of bacterial contamination10,11,12,13,14. Genomic sequencing from such small organisms requires a large number of animals and is prone to bacterial contamination; therefore, we have previously established a sequencing protocol using an ultralow input method starting from a single specimen of tardigrade, to minimize the risk of contaminations15. Using these data, we have further conducted a high-quality resequencing and reassembly of the genome of H. dujardini16,17.&#160;Here we describe in detail this method for genomic sequencing from a single tardigrade individual (Figure 1). The validation of this sequencing method is beyond the focus of this paper and has already been thoroughly discussed in our previous report16.
This method is comprised of two parts: the isolation of a single tardigrade with the lowest contamination possible, and the high-quality extraction of pictogram levels of DNA. The tardigrade is starved and rinsed thoroughly with water, as well as antibiotics, and observed under a microscope with 500X magnification to ensure the removal of any bacterial contamination. Previous estimates and measurements show that a single individual of tardigrade contains approximately 50 - 200 pg of genomic DNA16, which is extracted by cracking the chitin exoskeleton by freeze-thaw cycles or by manual homogenization. This genomic DNA is submitted to library construction and sequenced on a DNA sequencing instrument. An additional informatics analysis shows high-quality sequencing, as well as low levels of contamination in comparison to previous tardigrade sequencing projects.

<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

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.

Contaminant Exclusion:
This protocol involves a thorough washing of the tardigrade and a sterilization with antibiotics treatment to minimize contamination. It also involves a visual checking process to ensure the completeness of these processes. A microscope image made during the validation (step 2.4 of the protocol) is shown in Figure 2. When observed at a 500X magnification, bact...

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

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.

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.

ultralow-input-genome-sequencing-library-preparation-from-single

Research

JoVE Journal

Genetics

8.5K ビュー.  Keio University. 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.ele...