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 Preparation and Contaminant Exclusion
<ol>
	<li>Collect a single tardigrade, place it on the prepared agar plate, and wash it 2x - 3x with DW to remove any remaining particles.</li>
	<li>Incubate the tardigrade at 18 - 22 &#176;C for 24 h to remove any excess food from the intestines.</li>
	<li>Place the starved tardigrade in penicillin/streptomycin antibiotics for 2 - 6 h to remove any bacterial contamination and place the decontaminated animal on a clean slide glass using a P10 pipette.</li>
	<li>Observe the tardigrade under a microscope at 500X magnification and confirm that there are no remaining bacteria. 
	NOTE: The high magnification with a stereomicroscope is optimal, but an optical microscope may be used alternatively without applying a coverslip.</li>
	<li>Collect the individual using a P10 pipette with a maximum of 5 &#181;L of liquid, place it into a low-binding polymerase chain reaction (PCR) tube, and remove as much excess liquid as possible. 
	NOTE: The microtubes, PCR tubes, and pipette tips should all be low binding to minimize the loss of DNA. 
	NOTE: Conduct a homogenization as soon as possible, for the rapid desiccation or death of the animal can significantly damage the genomic DNA.</li>
</ol>
3. Homogenization and DNA Extraction
<ol>
	<li>Homogenize the animal to obtain its genomic DNA (gDNA) with one of the following methods. 
	NOTE: It is critical to use the specified kit shown in the Table of Materials in the following DNA extraction steps, for other kits (either column and bead based) are not effective at this extremely low input. The genomic lysis buffer has been supplied with 0.5% beta-mercaptoethanol prior to use.
	<ol>
		<li>Homogenize the animal with freeze-and-thaw cycles18.
		<ol>
			<li>Immediately following step 2.5, add 100 &#181;L of lysis buffer to the PCR tube containing the tardigrade.</li>
			<li>Place the PCR tube in liquid nitrogen for 10 min and, for 10 min, move it to a heat block warmed to 37 &#176;C. Repeat this step 2x. 
			NOTE: This step can be repeated when the homogenization is not sufficient or can be performed after the manual crushing.</li>
		</ol>
		</li>
		<li>Manually crush the animal.
		<ol>
			<li>Immediately following step 2.5, under a stereomicroscope, crush the individual with the P10 tip of the pipette by pressing the animal against the PCR tube wall, and immediately add 100 &#181;L of lysis buffer. 
			NOTE: It is critical to observe this procedure under a stereomicroscope, because the tardigrade can easily slip away, and inefficient crushing will result in a failure to extract gDNA. Make sure that the tardigrade cuticle is broken so that the lysis buffer can infiltrate the organism.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubate the tube for 30 min at room temperature for lysis to occur. 
	NOTE: A minimum of 30 min is required for an efficient lysis, but the incubation can be longer.</li>
	<li>Transfer the complete volume (100 &#181;L) of the lysis mixture to a clean 1.5-mL low-binding microtube.</li>
	<li>Add 100 &#181;L of lysis buffer to the low-binding PCR tube that was used for the homogenization and is now empty, and after pipetting the mixture, transfer it to the 1.5-mL low-binding microtube used in step 3.3. Repeat this step 2x.</li>
	<li>As in step 3.4, add 300 &#181;L of lysis buffer to the low-binding PCR tube, and, after pipetting, move the mixture to the 1.5-mL low-binding microtube. The 1.5-mL low-binding microtube should contain 600 &#181;L of the mixture after this step. 
	NOTE: These steps are to minimize the loss of gDNA bound to the PCR tube wall, by washing the sample multiple times.</li>
	<li>Add the total of 600 &#181;L of lysis mixture to the spin column placed in a collection tube and centrifuge it at 10,000 x g for 1 min.</li>
	<li>Re-apply the flow through to the column and centrifuge it at 10,000 x g for 1 min. 
	NOTE: This step is critical to ensure that most of the gDNA is bound to the column.</li>
	<li>Add 500 &#181;L of wash buffer to the spin column and centrifuge it at 10,000 x g for 1 min. Transfer the spin column to a clean 1.5-mL microtube.</li>
	<li>Apply 20 &#181;L of 10 mM Tris-HCl, pH 8.5, to the spin column, wait for 5 min at room temperature, and centrifuge it at 10,000 x g for 1 min. 
	NOTE: The elution buffer must not contain EDTA, for it interferes with the library preparation enzymes.</li>
	<li>Re-apply the flow through the spin column, and after 5 min of incubation at room temperature, centrifuge it for 1 min at 10,000 x g. 
	NOTE: This step is critical to ensure the maximal elution of the gDNA bound to the column.</li>
</ol>
4. Library Construction Sequence
<ol>
	<li>DNA fragmentation
	<ol>
		<li>Transfer 15 &#181;L of the gDNA eluant to a 15-&#181;L microtube for DNA fragmentation and centrifuge the tube for 1 min using a tabletop microcentrifuge. 
		NOTE: The specified microtube shown in the Table of Materials is optimal for a low input. The fragmentation with low-volume sonication is critical, and this cannot be substituted with enzymatic fragmentation because of the extremely low concentration of DNA.</li>
		<li>Fragment the gDNA to 550 bp. 
		NOTE: The 550 bp settings we used are as follows: peak incident power = 30 W, duty factor = 20%, cycles per burst = 50, treatment time = 23 s.</li>
		<li>After a thorough pipetting, transfer 10 &#181;L of the fragmented DNA mixture to a clean low-binding PCR tube. 
		NOTE: The experiment can be stopped here. Preserve the DNA at 4 &#176;C or -20 &#176;C.</li>
	</ol>
	</li>
	<li>Library construction sequence 
	NOTE: It is absolutely critical to use the specified kit in the Table of Materials in the following procedures, due to the low amount of input DNA.
	<ol>
		<li>Add 2 &#181;L of the template preparation buffer and 1 &#181;L of the template preparation enzyme and mix them thoroughly with a pipette.</li>
		<li>Perform the template preparation reaction on a thermal cycler with the following conditions: 22 &#176;C for 25 min, 55 &#176;C for 20 min, 4 &#176;C hold, and a heated lid at 101 - 105 &#176;C. Once the reaction is completed, proceed to the next step.</li>
		<li>Add 1 &#181;L of the library synthesis buffer and 1 &#181;L of the library synthesis enzyme to the template preparation reaction product and incubate the mixture at 22 &#176;C for 40 min (with a 4 &#176;C hold). Once the reaction is completed, proceed to the next step.</li>
		<li>Add 30 &#181;L of the library amplification master mix (25 &#181;L of the library amplification buffer, 1 &#181;L of the library amplification enzyme, and 4 &#181;L of nuclease-free water) and 5 &#181;L of the indexing reagent.</li>
		<li>Mix everything thoroughly with a pipette and centrifuge the mixture briefly with a tabletop centrifuge.</li>
		<li>Perform a PCR with the conditions presented in Table 1.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Temperature</td>
			<td>Time</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>72 &#730;C</td>
			<td>3 minutes</td>
			<td></td>
		</tr>
		<tr>
			<td>85 &#730;C</td>
			<td>2 minutes</td>
			<td></td>
		</tr>
		<tr>
			<td>98 &#730;C</td>
			<td>2 minutes</td>
			<td></td>
		</tr>
		<tr>
			<td>98 &#730;C</td>
			<td>20 seconds</td>
			<td rowspan="3">4 Cycles</td>
		</tr>
		<tr>
			<td>67 &#730;C</td>
			<td>20 seconds</td>
		</tr>
		<tr>
			<td>72 &#730;C</td>
			<td>40 seconds</td>
		</tr>
		<tr>
			<td>98 &#730;C</td>
			<td>20 seconds</td>
			<td rowspan="2">16 Cycles</td>
		</tr>
		<tr>
			<td>72 &#730;C</td>
			<td>50 seconds</td>
		</tr>
		<tr>
			<td>4 &#730;C</td>
			<td>Hold</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: PCR conditions.
<ol start="3">
	<li>Purification of the PCR products 
	NOTE: This step can be substituted with other purification methods. If an alternative method is used, make sure to check for the resulting DNA purity using a spectrophotometer. Absorbance ratios of 260/280 and 260/230 must both be above 1.8.
	<ol>
		<li>Add 50 &#181;L of magnetic beads, pipette the solution 10x, and centrifuge it briefly with a tabletop microcentrifuge.</li>
		<li>Incubate the solution for 2 min at room temperature.</li>
		<li>Incubate it on a magnetic stand for 5 min or until the solution becomes completely clear, and remove the supernatant.</li>
		<li>Add 200 &#181;L of freshly prepared 80% ethanol to the low-binding PCR tube on a magnet stand, wait for 30 s, and remove the supernatant. Repeat this step 2x. Do not disturb the beads.</li>
		<li>Briefly centrifuge the low-binding PCR tube with a tabletop centrifuge and remove any excess ethanol on the magnetic stand. Air-dry the beads but avoid overdrying.</li>
		<li>Resuspend the beads with 15 &#181;L of 10 mM Tris-HCl, pH 8.5, thoroughly pipette the solution so that the magnetic beads are homogeneously distributed, incubate the tube for 2 min at room temperature, and after a brief centrifugation, incubate it on a magnetic stand for 2 min until the solution is clear. 
		NOTE: The elution buffer should not contain EDTA, for it interferes with the sequencing chemistry.</li>
		<li>Transfer the supernatant, without disturbing the pellet, to a new low-binding PCR tube. 
		NOTE: The experiment can be stopped here. Preserve the DNA at 4 &#176;C or at -20 &#176;C for long-time storage.</li>
	</ol>
	</li>
</ol>
5. Quality Check, Quantification, and Sequence of the DNA
NOTE: A quality check is not conducted prior to this step due to the low amount of DNA.
<ol>
	<li>Validation of the DNA library size distribution 
	NOTE: Other high-sensitive electrophoresis systems with a digital reporting of size distribution can be used.
	<ol>
		<li>Return the electrophoresis buffer reagent and gel cartridge (25 - 1,000 bp in range) to room temperature.</li>
		<li>Add 3 &#181;L of electrophoresis buffer reagent with 1 &#181;L of the sequencing library and mix them thoroughly for 1 min with a vortex, and briefly centrifuge them with a tabletop centrifuge.</li>
		<li>Conduct electrophoresis and validate the library size distribution with the associated software. The main fragment peak should be broadly ranging from about 300 to 1,000 bp.</li>
	</ol>
	</li>
	<li>DNA quantification 
	NOTE: Other fluorescence-based methods or quantitative PCR can also be used, but spectrophotometry should be avoided, as it is not accurate enough for quantifying sequencing libraries.
	<ol>
		<li>Add 796 &#181;L of solution buffer and 4 &#181;L of fluorescent reagent and mix them thoroughly. Dispense 190 &#181;L of the working solution to two assay tubes and 197 &#181;L to one assay tube.</li>
		<li>Add 10 &#181;L of standards with known concentrations of DNA to each assay tube containing 190 &#181;L of the working solution, and 3 &#181;L of the prepared library to the assay tube containing 197 &#181;L of the working solution.</li>
		<li>Vortex the tubes briefly and centrifuge them on a tabletop centrifuge. Quantify the DNA using a fluorometer with 3-&#181;L settings.</li>
	</ol>
	</li>
	<li>DNA library sequence 
	Note: The sequencing platform must be compatible with the specified library construction kit.
	<ol>
		<li>Prepare the sequencing library based on the manufacturer&#39;s protocol.</li>
		<li>Set the reagent cassette and flow cell into the sequencing instrument and enter the sequencing run information following the manufacturer&#39;s protocol.</li>
		<li>Run the sequencing. 
		NOTE: We have conducted two sequencing runs: one sample/run, as well as four samples multiplexed in one run.</li>
	</ol>
	</li>
</ol>
6. Computational Analysis
<ol>
	<li>Base-call and, if required, demultiplex the reads.</li>
	<li>Validate the quality of the sequence data with FastQC19. 
	NOTE: For a more in-depth validation of the obtained data, see our previous report16.</li>
</ol>

<ol>
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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. 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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 ...

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

Research

JoVE Journal

Genetics

8.6K Views.  Keio University. 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.

Video: Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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

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 interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

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

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

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

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

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

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 been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

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

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 a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

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

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#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

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

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

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

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