Name: collection and extraction of saliva dna for next generation sequencing
Uploaded: 2014-08-27T15:00:00.000+00:00
Duration: 6 min 58 s
Description: Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).

NOTE: Prior to providing saliva samples all subjects gave informed consent conforming to the guidelines for treatment of human subjects at Nationwide Children&#8217;s Hospital.
1. Saliva Collection and Storage
<ol>
	<li>Prior to saliva collection, ensure that the subject&#8217;s mouth is free of food or other foreign substances by having the subject rinse their mouth with water and avoiding eating or drinking for 30 min before collecting the sample.</li>
	<li>Open a 15 ml centrifuge tube with 2.5 ml of DNA stabilization buffer2 making sure to avoid touching the inside of the cap or tube, and have the subject spit 2.5 ml of saliva into the buffer solution. Note: Collecting more than 2.5 ml of saliva can lead to sample degradation from an insufficient ratio of sample to DNA stabilization buffer. Collecting too little saliva will reduce expected yields from the protocol. To evaluate collection volumes, use the numbered gradients on the side of the tube.</li>
	<li>Replace cap and mix by inversion until the mixture is homogenized. Vigorous shaking is not necessary. Store the samples at RT for short-term storage or 4 &#176;C for long-term storage (&#62;3 months).</li>
</ol>
2. Initial Preparations and Cell Lysis
<ol>
	<li>Prior to starting the extraction, heat a water bath to 37 &#176;C, and prepare an ice bucket. Three 15 ml conical centrifuge tubes will be needed for each extracted sample. The three tubes will be used to hold the cell and protein pellet, the final extracted gDNA, and the isopropanol and ethanol supernatants.</li>
	<li>Retrieve samples from storage, and invert samples several times then vortex at medium speed for 15 sec.</li>
	<li>Dispense 2.5 ml of sample into a clean 15 ml centrifuge tube, and add 5 ml of Cell Lysis Solution. Mix the sample 50 times by inversion, and incubate at RT for 30 min.</li>
</ol>
3. RNA Removal
<ol>
	<li>Add 40 &#956;l of RNase A Solution at 100 mg/ml, and incubate at 37 &#176;C for 15 min.</li>
	<li>Remove the sample from the 37 &#176;C water bath and cool on ice for 3 min.
	<ol>
		<li>After the RNase A incubation, increase the temperature of the water bath to 65 &#176;C for the DNA rehydration step of the protocol.</li>
	</ol>
	</li>
</ol>
4. Protein and Lipid Removal
<ol>
	<li>Add 50 &#956;l of Proteinase K Solution at 20 mg/ml, mix several times by inversion, and incubate at RT for a minimum of 30 min. Note: This is a possible pausing point for the protocol. After the addition of the Proteinase K Solution, the sample can be stored at 4 &#176;C until the extraction can be completed. Storage at 4 &#176;C for up to 24 hr was not shown to have a significant effect on the extraction yields or DNA quality. Long-term storage at this stage has not been evaluated.</li>
	<li>Add 1.7 ml of Protein Precipitation Solution, vortex vigorously for 20 sec at high speed, and place on ice for 10 min.</li>
	<li>Once the samples have cooled on ice for 10 min, centrifuge for 10 min at 3,000 x g and 4 &#176;C. The precipitated proteins must form a tight pellet to continue. If the pellet is not tight or the solution is still cloudy, the samples can be cooled on ice for 5 min more and centrifugation repeated. The samples must be kept on ice to ensure a tight pellet.</li>
</ol>
5. Isolation and Purification of gDNA
<ol>
	<li>Into a clean 15 ml centrifuge tube, pipet 5 ml of Isopropanol and 8 &#956;l of pure Glycogen Solution at 20 mg/ml.</li>
	<li>Pour the supernatant containing the gDNA from step 4.3 into the tube containing the Isopropanol and Glycogen Solution, leaving behind the precipitated protein pellet. Once the supernatant has been added, gently mix the sample 50 times by inversion and centrifuge for 30 min at 3,000 x g and 4 &#176;C.</li>
	<li>Pour the supernatant slowly into a clean 15 ml tube. After removal of the supernatant, add 1 ml of 70% ethanol to wash the pellet by slowly rocking and gently moving the ethanol over the precipitated pellet several times. Retain the ethanol in the tube.</li>
	<li>After the initial wash, centrifuge the sample for 1 min at 2,000 x g and 20 &#176;C. This centrifugation step can be done at either 4 &#176;C or 20 &#176;C. No significant effect of temperature has been shown for this step.</li>
	<li>Following the initial wash and centrifugation of the pellet, slowly pour the ethanol wash from the tube and discard, then perform a second wash by repeating steps 5.3 and 5.4.</li>
	<li>After the removal of the supernatant from the second wash, allow the pellet to air dry for 15 min.
	<ol>
		<li>If the sample has not completely dried, air dry for another 15 min.</li>
	</ol>
	</li>
</ol>
6. Rehydration of gDNA
<ol>
	<li>Once the sample has dried, add 300 &#956;l of Tris-EDTA to rehydrate the dried gDNA pellet.</li>
	<li>Vortex the sample for 5 sec&#160;at medium speed and place in a 65 &#176;C hot water bath for 1 hr.</li>
	<li>Remove the samples from the water bath and incubate O/N at RT. 
	NOTE: All products and reagents used are listed in the Materials Table, as well as Table 4.</li>
</ol>

<ol>
	<li>Quinque, D., Kittler, R., Kayser, M., Stoneking, M., Nasidze, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+saliva+as+a+source+of+human+DNA+for+population+and+association+studies.">Evaluation of saliva as a source of human DNA for population and association studies.</a> Analytical Biochemistry. 353, 272-277 (2006).</li><li>Min, J. L., 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=High+mircosatellite+and+SNP+genotyping+success+rates+established+in+a+large+number+of+genomic+DNA+samples+extracted+from+mouth+swabs+and+genotypes.">High mircosatellite and SNP genotyping success rates established in a large number of genomic DNA samples extracted from mouth swabs and genotypes.</a> Twin Research and Human Genetics. 9, 501-506 (2006).</li><li>Dlugos, D. J., Scattergood, T. M., Ferraro, T. N., Berrettinni, W. H., Buono, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+rates+and+fear+of+phlebotomy+in+pediatric+patients+in+a+genetic+study+of+epilepsy.">Recruitment rates and fear of phlebotomy in pediatric patients in a genetic study of epilepsy.</a> Epilepsy &amp; Behavior. 6, 444-446 (2005).</li><li>Etter, J. F., Neidhart, E., Bertand, S., Malafosse, A., Bertrand, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+saliva+by+mail+for+genetic+and+cotinine+analyses+in+participants+recruited+through+the+internet.">Collecting saliva by mail for genetic and cotinine analyses in participants recruited through the internet.</a> European Journal of Epidemiology. 20, 833-838 (2005).</li><li>Hansen, T. V., Simonsen, M. K., Nielsen, F. C., Hundrup, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+blood,+saliva,+and+buccal+cell+samples+in+a+pilot+study+on+the+danish+nurse+cohort:+Comparison+of+the+response+rate+and+quality+of+genomic+DNA.">Collection of blood, saliva, and buccal cell samples in a pilot study on the danish nurse cohort: Comparison of the response rate and quality of genomic DNA.</a> Cancer Epidemiol Biomarkers Prev. 16, 2072-2076 (2007).</li><li>Van Schie, R. C. A. A., Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva:+A+convenient+source+of+DNA+for+analysis+of+bi-allelic+polymorphisms+of+fc&#947;+receptor+iia+(cd32)+and+fc&#947;+receptor+iiib+(cd16).">Saliva: A convenient source of DNA for analysis of bi-allelic polymorphisms of fc&#947; receptor iia (cd32) and fc&#947; receptor iiib (cd16).</a> Journal of Immunological Methods. 208, 91-101 (1997).</li><li>Dawes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates,+from+salivary+analyses,+of+the+turnover+time+of+oral+mucosal+epithelium+in+humans+and+the+number+of+bacteria+in+an+edentulous+mouth.">Estimates, from salivary analyses, of the turnover time of oral mucosal epithelium in humans and the number of bacteria in an edentulous mouth.</a> Archives of Oral Biology. 48, 329-336 (2003).</li><li>Bahlo, M., 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=Saliva-derived+DNA+performs+well+in+large-scale,+high-density+single-nucleotide+polymorphism+microarray+studies.">Saliva-derived DNA performs well in large-scale, high-density single-nucleotide polymorphism microarray studies.</a> Cancer Epidemiol Biomarkers Prev. 19, 794-798 (2010).</li><li>Hu, 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=Genotyping+performance+between+saliva+and+blood-derived+genomic+dnas+on+the+dmet+array:+A+comparison.">Genotyping performance between saliva and blood-derived genomic dnas on the dmet array: A comparison.</a> PLoS ONE. 7 (3), e33968 (2012).</li><li>Simmons, T. R., 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=Increasing+genotype-phenotype+model+determinism:+Application+to+bivariate+reading/language+traits+and+epistatic+interactions+in+language-impaired+families.">Increasing genotype-phenotype model determinism: Application to bivariate reading/language traits and epistatic interactions in language-impaired families.</a> Human Heredity. 70, 232-244 (2010).</li><li>Zeugin, J. A., Hartley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+precipitation+of+DNA.">Ethanol precipitation of DNA.</a> Focus. 7, 1-2 (1985).</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, 1754-1760 (2009).</li><li>McKenna, A., 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+Genome+Analysis+Toolkit:+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data.">The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.</a> Genome Res. 20, 1297-1303 (2010).</li><li>DePristo, M., 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=A+framework+for+variation+discovery+and+genotyping+using+next-generation+DNA+sequencing+data.">A framework for variation discovery and genotyping using next-generation DNA sequencing data.</a> Nature Genetics. 43, 491-498 (2011).</li></ol>

The authors have nothing to disclose.

The present procedure is an optimized DNA extraction protocol that has considerably improved yield of high molecular weight DNA compared to standard methods, without compromising DNA quality. The critical step with the biggest effect on yield the most was step 5.2, which includes a longer centrifugation step during ethanol precipitation than any published protocol reviewed here, except one that was not widely distributed11. No changes in DNA quality associated with this longer centrifugation were detected, indicating that most of the available DNA from whole saliva collection is not degraded and high molecular weight.
Whole saliva collection has limitations in sample quality such as the potential for foreign contaminants, which needs to be minimized at the collection stage, and the presence of excessive protein in the sample that can be a sign of an underlying infection. Large amounts of protein or foreign contaminants can remain in the final extracted DNA thereby making quantification inaccurate. If there are residual proteins or contaminants after rehydration (step 6) are suspected, a sample clean up can be performed by starting at the protein precipitation step with reagent volumes scaled to reflect the sample input volume. Another limitation of the protocol is the length of time required to perform the steps. Multiple samples can be run in parallel; however, it is recommended that no more than 24 parallel extractions be run simultaneously. This is particularly important for the protein precipitation step, where the sample must remain cold to ensure a tight pellet and running more than 24 samples may allow pellets time to re-dissolve.
The protocol presented here is the most cost effective method considered (see Table 3). While this protocol does use reagents from the Puregene extraction kit, a smaller volume than recommended in the Puregene protocol is used without compromising extraction yield and it is this reduction in reagents that drives the cost savings relative to the protocol. Note that the calculated cost for extraction uses the list price for each item and does not reflect any discounts. The cost per extraction with the optimized protocol can be reduced further with bulk orders or discounts through company sales representatives.
Evidence has also been provided that this protocol is suitable for use with next generation sequencing. The data obtained provides further evidence of the utility of saliva samples for human genetics research in many diseases where blood is not routinely available. While blood and cell line derived DNA continues to be the preferred source of genetic material for testing, whole saliva collection is a viable alternative when such sources are not available, when patient enrollment is affected by their collection or phlebotomy is not available or impractical.

Obtaining high quality DNA for human genetic studies is essential in the disease gene discovery process. Blood, though requiring an invasive procedure and also being more expensive than saliva collection, is favored for creating immortalized cell lines as an infinite source of DNA, or iPSCs for functional studies, and sometimes blood DNA is used when cell lines are not available. However, obtaining blood requires a trained phlebotomist and blood has a shorter half-life than saliva1. DNA from saliva is less expensive and easier to obtain, since it can be collected and sent through the mail without the need for a phlebotomist, thereby increasing potential subject pools well beyond the catchment area of hospitals and laboratories2. Study enrollment may be improved when subjects have the option of giving a saliva sample instead of blood3, 4. Concerns about the quantity and quality of DNA from saliva may have limited its widespread use despite numerous studies recent studies showing the suitability of whole saliva, with an average of 4.3 x 105 cells per milliliter, for DNA testing over the older buccal swabs methods that did not obtain significant amounts of saliva2, 3, 4, 5, 6. While a modest literature exists showing the suitability of whole saliva derived DNA for genotyping applications including microarray-based methods8, 9, 10, no studies have examined next generation sequencing (NGS). The goal for optimizing this whole saliva DNA extraction protocol was to maximize quantity and quality for genetics applications in a cost effective way that is easily implemented in laboratories with common reagents and consumables.
DNA extraction from saliva requires several procedures: 1) collection and storage, 2) cell lysis, 3) RNase treatment, 4) protein precipitation, 5) ethanol precipitation, 6) DNA rehydration. The DNA Stabilization Buffer solution, described previously2, functions adequately without alteration. No attempt to optimize the RNase treatment and DNA rehydration steps was made. For each remaining step, several variables that could affect yield were identified. Each variable was manipulated individually and improvement in yield and quality was assessed statistically. For variables that were shown to improve yield and/or DNA quality, the optimal values were included in the final protocol.

<table><tbody><tr><td>15 ml Centrifuge Tubes</td><td>Fisher</td><td>12-565-268</td><td></td></tr><tr><td>Cell Lysis Solution</td><td>Qiagen</td><td>158908</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td></td></tr><tr><td>Protein Precipitation Solution</td><td>Qiagen</td><td>158912</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher</td><td>A416-4</td><td></td></tr><tr><td>Glycogen</td><td>EZ-BioResearch</td><td>S1003</td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher</td><td>04-355-305</td><td></td></tr><tr><td>Tris-EDTA (TE)</td><td>Fisher</td><td>BP2473-1</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>AC194090010</td><td></td></tr><tr><td>Tris HCl</td><td>Fisher</td><td>BP1757-100</td><td></td></tr><tr><td>EDTA (0.5 M) Solution</td><td>Fisher</td><td>03-500-506</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Fisher</td><td>BP166-100&amp;nbsp;</td><td></td></tr><tr><td>Analog Vortex Mixer</td><td>Fisher</td><td>02-215-365</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>5811 000.010</td><td></td></tr></tbody></table>

collection and extraction of saliva dna for next generation sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

To determine optimal parameters for DNA extraction a series of paired DNA extractions was performed. A single saliva sample was split and each portion tested with one of two possible values for a given variable. At least eight replicates of each paired test were performed (e.g., a single saliva sample was aliquoted to test extraction both with and without initial 50 &#176;C incubation). Optimization was based on four standard metrics: total DNA yield, the 260/280 value, the 260/230 value, and visual inspection of electrophoresed DNA to assess fragmentation. Not all possible combinations of the variables were assessed statistical interactions (N=169 combinations), opting instead to assess the marginal effect of each variable individually. Effects were tested using a multi-way repeated-measures ANOVA and estimated effects were derived from the equivalent regression equation. All significant effects are summarized in Table 1, shown as average change in yield (ng/&#181;l per ml of saliva input) or DNA quality (260/280 and 260/230).
Cell lysis (step 2) was optimized by assessing: 1) the presence/absence of a 50 &#176;C incubation (1 hr) prior to cell lysis to ensure that Proteinase K degradation and cell lysis mediated by the storage buffer went to completion, 2) presence/absence of a homogenization by vortexing step (medium speed, 15 sec), and 3) lysis solution incubation time (5 versus 30 min). The 30 min cell lysis incubation increased yield by an average of 3.5% (p&#60;.01) but no other cell lysis variable had a significant effect on yield. Vortexing decreased the 260/280 ratio by a statistically significant (p&#60;.001) but practically small 0.03.
Protein precipitation (step 4) is preceded by Proteinase K digestion to disrupt amino acid chains, improving protein precipitation efficiency and releasing captured DNA. The amount of Proteinase K was varied ten-fold. Centrifugation temperature was reduced from 20 &#176;C to 4 &#176;C. Increasing the amount of Proteinase K caused a statistically significant decrease in yield (8.7%) and also slightly improved both the 260/280 and 260/230 ratios.
Ethanol precipitation (step 5) was the last stage of the protocol examined. The amount of glycogen carrier (0, 8 &#181;l) was varied, as was the total centrifugation time (5 vs. 30 min11). Only centrifugation time significantly affected yield, with an average increase of 290%. The longer spin also decreased the 260/280 ratio slightly (0.05). No significant effect of glycogen on yield was observed during the experiments; though the total quantity of DNA in these extractions was sufficiently large that glycogen would not typically be used. Despite the lack of effect in these samples, it is still recommend to use glycogen to minimize the risk of reduced yields whenever saliva input volume is lower than given here or if there is any other reason to believe yield will be low.
Visual inspection of the representative DNA samples (Figure 1) indicated that the extracted DNA was not greatly fragmented for any saliva DNA extraction procedure, but rather showed an appropriate high molecular weight band without the smearing indicative of degraded DNA. After RNase A digestion, the protocol produced an average 260/280 of 1.74.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51697/51697fig1highres.jpg" width="500" /> 
Figure 1.&#160;Quality of saliva derived DNA. Four extraction procedures were applied to the same saliva collection. (A) Samples were electrophoresed on a 0.8% agarose gel (250 ng DNA). All variations of the saliva DNA extraction protocols result in high molecular weight (&#62;20 kb) DNA, with no evidence of degradation. Lane: 1 DNA ladder, 2 &#38; 3 Oragene prepIT L2P Protocol samples, 4 &#38; 5 Gentra Puregene Body Fluids Protocol, 6 &#38; 7 the optimized protocol without RNA removal step, 8 &#38; 9 the optimized protocol with the RNA remove step. Lanes 2-7 are directly analogous protocols on the same saliva samples. Lanes 8 &#38; 9 show that the RNA removal step does not introduce DNA degradation. (B) Samples were electrophoresed on a 2% agarose gel (150 ng DNA). A slight RNA peak is observable near the bottom of the gel in lanes 2 through 7 (conventions as above). Lanes 8 &#38; 9 show the effectiveness of the RNA removal step.
The RNA Removal Step (step 3 with RNase A) is critical for accurate quantification of DNA. During testing, consistently high RNA content was observed, as determined by the ratio of double stranded DNA to RNA measured by a Qubit 2.0 Fluorometer. On average, nucleic acid content from samples without RNase A treatment consisted of 46.6% (&#177;0.4) RNA. Samples that underwent the RNA Removal Step read as &#8220;&#60;20 ng/ml&#8221;, which is the lowest possible reading for the Qubit&#8217;s RNA detection.
The DNA obtained through this optimized protocol was of sufficient quality for high throughput sequencing when the additional RNase A step was applied. To attain targeted resequencing data, a custom Agilent SureSelect Target Enrichment kit was applied to 24 samples, targeting 2.6 Mb of sequence. High throughput sequencing was conducted on 12 barcoded (indexed) samples per lane. Sequence reads were BWA-aligned to the hg19 reference genome12, then application of GATK13 base quality score recalibration, indel realignment, duplicate removal, SNP discovery and genotyping simultaneously across all 24 samples was performed using the best practice hard filtering parameter values14. All 24 samples yielded high quality NGS data (Table 2). Of reads that passed Illumina&#8217;s standard filters and had Q&#62;20, 91.4% aligned to the sequence enrichment target regions, providing an average on-target coverage depth of &#62;30x coverage at Q&#62;100, well within the necessary limits for rare SNP discovery in each sample. The average strand balance was 49.9%. Comparing variant calls with Illumina microarray genotypes yielded a concordance of 98.9%.
<table border="0" cellpadding="0" cellspacing="0" width="352">
	<tbody>
		<tr>
			<td>Candidate Variable</td>
			<td>ng/&#181;l&#160;</td>
			<td>260/280</td>
			<td>260/230</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>n.s.</td>
			<td>-0.03***</td>
			<td>n.s.</td>
		</tr>
		<tr>
			<td>x30 min Cell Lysis Incubation</td>
			<td>3.5%**</td>
			<td>n.s.</td>
			<td>n.s.</td>
		</tr>
		<tr>
			<td>Proteinase K x10&#160;</td>
			<td>-8.7%*</td>
			<td>-0.05***</td>
			<td>-0.03***</td>
		</tr>
		<tr>
			<td>30 min spin</td>
			<td>290.2%***</td>
			<td>-0.05***</td>
			<td>n.s.</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>n.s.</td>
			<td>n.s.</td>
			<td>-0.37***</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Effect Size of Optimized Variable on Quantity/Quality Metrics. All effect sizes are in the units listed in the column header. p-values from ANOVA: *p&#60;.05; **p&#60;.01; ***p&#60;.001; n.s. not significant
<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr>
			<td>Quality Metric</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Target length</td>
			<td>1,708 kb</td>
		</tr>
		<tr>
			<td>Target Covered &#62; 30x</td>
			<td>85.22%</td>
		</tr>
		<tr>
			<td>SNPs in dbSNP</td>
			<td>92.55%</td>
		</tr>
		<tr>
			<td>Array agreement</td>
			<td>98.99%</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Quality of High Throughput Sequence from Target Enrichment.
<table border="0" cellpadding="0" cellspacing="0" width="668">
	<tbody>
		<tr>
			<td rowspan="14">New Optimized Protocol</td>
			<td>Item</td>
			<td>Distributer</td>
			<td>Catalog #</td>
			<td>Purchasing Unit</td>
			<td>Cost/Unit</td>
			<td>Cost/Collection</td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>12-565-268</td>
			<td>500 Tubes</td>
			<td>$233.50</td>
			<td>$3.2690</td>
		</tr>
		<tr>
			<td>Cell Lysis Solution</td>
			<td>Qiagen</td>
			<td>158908</td>
			<td>1 L</td>
			<td>$401.00</td>
			<td>$3.2080</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma</td>
			<td>P6556</td>
			<td>1 g</td>
			<td>$713.00</td>
			<td>$1.5686</td>
		</tr>
		<tr>
			<td>Protein Precipitation Solution</td>
			<td>Qiagen</td>
			<td>158912</td>
			<td>350 ml</td>
			<td>$350.00</td>
			<td>$2.7200</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>A416-4</td>
			<td>Case of 4 x 4 L</td>
			<td>$486.71</td>
			<td>$0.2434</td>
		</tr>
		<tr>
			<td>Glycogen Solution (20 mg/ml)</td>
			<td>EZ-BioResearch</td>
			<td>S1003</td>
			<td>1 ml</td>
			<td>$51.00</td>
			<td>$0.8160</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td>Case of 4 x 1 gal.</td>
			<td>$123.03</td>
			<td>$0.0325</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE)</td>
			<td>Fisher</td>
			<td>BP2473-1</td>
			<td>1 L</td>
			<td>$68.67</td>
			<td>$0.0412</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>AC194090010</td>
			<td>1 kg</td>
			<td>$34.65</td>
			<td>$0.000004</td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Fisher</td>
			<td>BP1757-100</td>
			<td>100 ml</td>
			<td>$57.84</td>
			<td>$0.0116</td>
		</tr>
		<tr>
			<td>EDTA(0.5 M) Solution</td>
			<td>Fisher</td>
			<td>03-500-506</td>
			<td>100 ml</td>
			<td>$33.60</td>
			<td>$0.0134</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100&#160;</td>
			<td>100 g</td>
			<td>$59.95</td>
			<td>$0.0060</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Total Cost</td>
			<td>$11.93</td>
		</tr>
		<tr>
			<td rowspan="8">Oragene</td>
			<td>Item</td>
			<td>Distributer</td>
			<td>Catalog #</td>
			<td>Purchasing Unit</td>
			<td>Cost/Unit</td>
			<td>Cost/Collection</td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>12-565-268</td>
			<td>500 Tubes</td>
			<td>$233.50</td>
			<td>$0.9340</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-100</td>
			<td>100 ml</td>
			<td>$34.29</td>
			<td>$1.6459</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td>Case of 4 x 1 gal.</td>
			<td>$123.03</td>
			<td>$0.0081</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE)</td>
			<td>Fisher</td>
			<td>BP2473-1</td>
			<td>1 L</td>
			<td>$68.67</td>
			<td>$0.0687</td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Genesee</td>
			<td>22-281A</td>
			<td>500 Tubes</td>
			<td>$22.85</td>
			<td>$0.0457</td>
		</tr>
		<tr>
			<td>Oragene Collection KIT</td>
			<td>Oragene</td>
			<td>OG-500</td>
			<td>1</td>
			<td>$25.00</td>
			<td>$25.00</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Total Cost</td>
			<td>$27.70</td>
		</tr>
		<tr>
			<td rowspan="14">Puregene</td>
			<td>Item</td>
			<td>Distributer</td>
			<td>Catalog #</td>
			<td>Purchasing Unit</td>
			<td>Cost/Unit</td>
			<td>Cost/Collection</td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>12-565-268</td>
			<td>500 Tubes</td>
			<td>$233.50</td>
			<td>$0.9340</td>
		</tr>
		<tr>
			<td>Cell Lysis Solution</td>
			<td>Qiagen</td>
			<td>158908</td>
			<td>1 L</td>
			<td>$401.00</td>
			<td>$4.0100</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Qiagen</td>
			<td>158918</td>
			<td>650 ml</td>
			<td>$73.10</td>
			<td>$7.8723</td>
		</tr>
		<tr>
			<td>Protein Precipitation Solution</td>
			<td>Qiagen</td>
			<td>158912</td>
			<td>350 ml</td>
			<td>$350.00</td>
			<td>$4.0000</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher</td>
			<td>A4164</td>
			<td>Case of 4 x 4 L</td>
			<td>$486.71</td>
			<td>$0.3650</td>
		</tr>
		<tr>
			<td>Glycogen Solution</td>
			<td>Qiagen</td>
			<td>158930</td>
			<td>500 ml</td>
			<td>$64.30</td>
			<td>$2.5720</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td>Case of 4 x 1 gal.</td>
			<td>$123.03</td>
			<td>$0.0975</td>
		</tr>
		<tr>
			<td>DNA Hydration Solution</td>
			<td>Qiagen</td>
			<td>158914</td>
			<td>100 ml</td>
			<td>$69.80</td>
			<td>$0.1396</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>AC19409-0010</td>
			<td>1 kg</td>
			<td>$34.65</td>
			<td>$0.000004</td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Fisher</td>
			<td>BP1757-100</td>
			<td>100 ml</td>
			<td>$57.84</td>
			<td>$0.0116</td>
		</tr>
		<tr>
			<td>EDTA(0.5 M) Solution</td>
			<td>Fisher</td>
			<td>03-500-506</td>
			<td>100 ml</td>
			<td>$33.60</td>
			<td>$0.0134</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100&#160;</td>
			<td>100 g</td>
			<td>$59.95</td>
			<td>$0.0060</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Total Cost</td>
			<td>$19.99</td>
		</tr>
	</tbody>
</table>
Table 3.&#160;Cost comparison of the optimized protocol to extract DNA from 2 ml of whole saliva with other commercially available protocols. All reagents and consumables required for DNA extraction have been assessed using standard list prices available on the internet as of September 30, 2013. Note that this protocol is written to extract DNA from 1.25 ml of whole saliva (i.e., 2.5 ml of saliva and buffer, see step 2.3) as this value represents half of the total volume in the saliva collection tube. For the cost comparison, 2 ml was chosen as this is the amount associated with a common commercially available kit (Oragene).
<table border="0" cellpadding="0" cellspacing="0" width="264">
	<tbody>
		<tr>
			<td colspan="3">DNA Stabilization Buffer (250 ml)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (ml)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>1 M NaCl</td>
			<td>1.461 g</td>
			<td>0.1 M</td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>2.5</td>
			<td>0.01 M</td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>5</td>
			<td>0.01 M</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>12.5</td>
			<td>0.014 M</td>
		</tr>
		<tr>
			<td>Proteinase K Solution (20 mg/ml)</td>
			<td>2.5</td>
			<td>6.92x10-6 M</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>227.5</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Proteinase K Solution (20 mg/ml)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (ml)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>Proteinase K powder</td>
			<td>500 mg</td>
			<td>6.92x10-4 M</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>25</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">70% Ethanol (500 ml)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (ml)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>Ethanol 95%</td>
			<td>368.5</td>
			<td>12.63 M</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>131.5</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4.&#160;Recipes for Reagents.

Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

Collection and Extraction of Saliva DNA for Next Generation Sequencing

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

collection-and-extraction-of-saliva-dna-for-next-generation-sequencing

Universidad San Sebastian

Research

JoVE Journal

Medicine

39.1K Views.  The Research Institute at Nationwide Children's Hospital. DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Video: Collection and Extraction of Saliva DNA for Next Generation Sequencing

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

Genetics

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

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Cancer Research

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

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 ...

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

The protocol is intended to serve as a blueprint for universities and other organizations considering large-scale testing for SARS-CoV-2 or developing preparedness plans for future viral outbreaks.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Biology

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

Podsumowanie

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Metagenomic Analysis of Silage

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

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

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

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Targeted DNA Methylation Analysis by Next-generation Sequencing