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/pubmed?term=((Evaluation+of+saliva+as+a+source+of+human+DNA+for+population+and+association+studies%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((High+mircosatellite+and+SNP+genotyping+success+rates+established+in+a+large+number+of+genomic+DNA+samples+extracted+from+mouth+swabs+and+genotypes%5BTitle%5D)+AND+%22Twin+Research+and+Human+Genetics%22%5BJournal%5D)">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/pubmed?term=((Recruitment+rates+and+fear+of+phlebotomy+in+pediatric+patients+in+a+genetic+study+of+epilepsy%5BTitle%5D)+AND+%22Epilepsy+%26+Behavior%22%5BJournal%5D)">Recruitment rates and fear of phlebotomy in pediatric patients in a genetic study of epilepsy.</a> Epilepsy & 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/pubmed?term=((Collecting+saliva+by+mail+for+genetic+and+cotinine+analyses+in+participants+recruited+through+the+internet%5BTitle%5D)+AND+%22European+Journal+of+Epidemiology%22%5BJournal%5D)">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/pubmed?term=((Collection+of+blood%2C+saliva%2C+and+buccal+cell+samples+in+a+pilot+study+on+the+danish+nurse+cohort%3A+Comparison+of+the+response+rate+and+quality+of+genomic+DNA%5BTitle%5D)+AND+%22Cancer+Epidemiol+Biomarkers+Prev%22%5BJournal%5D)">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/pubmed?term=((Saliva%3A+A+convenient+source+of+DNA+for+analysis+of+bi-allelic+polymorphisms+of+fc%CE%B3+receptor+iia+%28cd32%29+and+fc%CE%B3+receptor+iiib+%28cd16%29%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Saliva: A convenient source of DNA for analysis of bi-allelic polymorphisms of fcγ receptor iia (cd32) and fcγ 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/pubmed?term=((Estimates%2C+from+salivary+analyses%2C+of+the+turnover+time+of+oral+mucosal+epithelium+in+humans+and+the+number+of+bacteria+in+an+edentulous+mouth%5BTitle%5D)+AND+%22Archives+of+Oral+Biology%22%5BJournal%5D)">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/pubmed?term=((Saliva-derived+DNA+performs+well+in+large-scale%2C+high-density+single-nucleotide+polymorphism+microarray+studies%5BTitle%5D)+AND+%22Cancer+Epidemiol+Biomarkers+Prev%22%5BJournal%5D)">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/pubmed?term=((Genotyping+performance+between+saliva+and+blood-derived+genomic+dnas+on+the+dmet+array%3A+A+comparison%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Increasing+genotype-phenotype+model+determinism%3A+Application+to+bivariate+reading%2Flanguage+traits+and+epistatic+interactions+in+language-impaired+families%5BTitle%5D)+AND+%22Human+Heredity%22%5BJournal%5D)">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/pubmed?term=((Ethanol+precipitation+of+DNA%5BTitle%5D)+AND+%22Focus%22%5BJournal%5D)">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/pubmed?term=((Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+Transform%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((The+Genome+Analysis+Toolkit%3A+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">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/pubmed?term=((A+framework+for+variation+discovery+and+genotyping+using+next-generation+DNA+sequencing+data%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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, ind...

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 o...

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

Research

JoVE Journal

Medicine

39.2K 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 ...

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 ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Bioengineering

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. ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Immunology and Infection

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

Summary

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Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

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