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>

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

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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

9.5K Views.  Keio University. Besmetting tijdens de genomic sequencing van microscopische organismen blijft een groot probleem. Hier, laten we een methode om het genoom van een beerdiertjes uit een enkel exemplaar met zo weinig als 50 pg van genomic DNA zonder het hele genoom versterking om te minimaliseren van het risico van besmetting.

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

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

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

Environment

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 ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biochemistry

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology. Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to error-prone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination.

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data ...

A Comprehensive Approach to Studying the Promoter Interactome in Scarce Cells

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Author Spotlight: An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Bioengineering

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Neuroscience

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

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