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

注意:在此之前提供唾液样本所有受试者知情同意书符合指引处理在全国儿童医院人类受试者。 1，唾液的采集和存储<ol><li>此前唾液的收集，确保拍摄对象的嘴是免费的食物或其他异物所具有的主题冲洗他们的嘴的水，避免进食或收集样本前，喝了30分钟。 </li><li>用2.5毫升的DNA稳定缓冲液2，确保避免触及上限或管内打开一个15毫升的离心管中，并有课题吐唾液2.5毫升放入缓冲液。注:收集多个2.5毫升唾液可导致样品降解从样品的DNA的稳定缓冲液的不足比率。收集太少唾液会减少从协议预期产量。为了评估珍藏册，使用编号为梯度对T的侧面他管的。 </li><li>更换盖和通过反转混合，直到混合物均化。剧烈摇动是没有必要的。储存在室温下将样品用于短期存储或4℃下长期储存（&gt; 3个月）。 </li></ol> 2，前期准备和细胞裂解<ol><li>在此之前开始开采，加热水浴至37℃，并准备一个冰桶。三的15毫升锥形离心管中，将需要为每个提取的样品。三管将被用于保持细胞和蛋白质沉淀，最终提取基因组DNA，并在异丙醇和乙醇上清液。 </li><li>从存储器中检索的样品，并翻转试样几次然后涡流在中等速度持续15秒。 </li><li>免除2.5毫升样品放入一个干净的15ml离心管中，加入5毫升细胞裂解液。通过倒置混合样品50次，并在室温下孵育30分钟。 </li></ol> 3，RNA的去除<ol><li>加入40μl的RNase溶胶ution在100毫克/毫升，并在37℃下进行15分钟。 </li><li>从37℃水浴中取出样品，并在冰上冷却3分钟。 <ol><li>的RNA酶A孵育后，提高水浴温度至65℃的协议的DNA再水合步骤。 </li></ol></li></ol> 4，蛋白质和脂肪去除<ol><li>加入50微升蛋白酶K溶液20毫克/毫升，颠倒混匀数次，并在室温下孵育至少30分钟。注:这是一个可能的暂停点的协议。在加入蛋白酶K溶液中后，可将样品储存于4℃直至萃取就可以完成。储存在4℃下长达24小时未显示出对萃取率或DNA质量显著效果。长期存放在这个阶段并没有进行评估。 </li><li>添加1.7毫升的蛋白沉淀溶液，涡旋混合20秒，在高速，并置于冰上10分钟。 </lI&gt; <li>一旦样品已在冰上冷却10分钟，离心以3,000 XG 10分钟，4℃。沉淀的蛋白质必须形成一个紧密的小球继续。如果沉淀不紧或溶液仍是浑浊的样品可以在冰上冷却5分钟以上，并离心分离重复进行。样品必须保持在冰上，以确保紧密的颗粒。 </li></ol> 5，分离和基因组DNA纯化<ol><li>到一个干净的15ml离心管，移液管5毫升异丙醇和8微升纯糖原溶液20毫克/毫升。 </li><li>倾从步骤4.3到含有异丙醇和糖原溶液的管含有基因组DNA的上清液，剩下的沉淀的蛋白质沉淀物的后面。一旦上清液已经加入，轻轻在3000 XG和4℃下混匀50次颠倒和离心30分钟。 </li><li>慢慢倒入上清到一个干净的15ml试管中。除去上清液后，加入1 ml 70％的乙醇，以通过缓慢摇动，然后轻轻移动的乙醇在沉淀沉淀数次洗涤沉淀。保留在管中的乙醇。 </li><li>初始洗涤后，离心样品在2000 XG 1分钟和20℃。此离心步骤可以在任4°C或20°C下进行。温度的无显著效果已被证实对于此步骤。 </li><li>以下将沉淀的初始洗涤并离心，慢慢倾从管的乙醇洗涤并弃去，然后通过重复步骤5.3和5.4进行第二次洗涤。 </li><li>除去来自第二洗涤上清液后，使沉淀物在空气中干燥15分钟。 <ol><li>如果样品没有完全干燥，风干另外15分钟。 </li></ol></li></ol> 6补液的基因组DNA <ol><li>一旦样品已干燥，加入300微升的Tris-EDTA至再水化干燥的gDNA粒料。 </li><li>涡样品5秒，中速和发生在65℃的热水浴中搅拌1小时。 </li><li>从水浴中取出样品，并培育在o / n RT。 注意:所有的产品和试剂使用中列出的材料表，以及表4中 。 </li></ol>

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The authors have nothing to disclose.

本程序是一个优化的DNA提取的协议，已大大改善了高分子量DNA的产量相比，标准的方法，而不损害DNA的质量。与对产量的大部分影响最大的关键步骤是5.2步，其中包括在乙醇沉淀比任何出版协议审查这里，除了一个没有广泛分布11较长的离心步骤。检测DNA的质量没有改变与该较长的离心关联，表明大部分从唾液采集的可用的DNA是不是退化和高的分子量。 唾液收集在样?...

获得高质量的DNA对人类遗传研究是在疾病基因的发现进程至关重要。血，虽然需要侵入性手术，也比唾液采集更昂贵，是有利的，用于创建永生化细胞系的DNA的无限源，或iPS细胞的功能性研究，有时血液的DNA时使用的细胞系是不可用的。然而，获得的血液需要经过培训的采血和血液有一个较短的半衰期比口水1。从唾液DNA是较便宜，容易得到，因为它可以被收集，并通过邮件发送，而不需要用于抽血，由此增加潜在受试者池远远超出医院和实验室2的集水面积。当受试者具有给人一种唾液样品代替血液3,4的选择研究登记可以得到改进。左右的数量，从唾液DNA的质量问题可能已限制了其广泛我们ë尽管许多研究最近的研究显示全唾液的适用性，平均每毫升4.3×10 5个细胞，用于对较早的口腔拭子的方法未获得显著量唾液2，3，4，5的DNA检测， 6，虽然适度的文学存在呈现出唾液的DNA是否适合进行基因分型应用，包括基于微阵列的方法，8，9，10，没有研究探讨了新一代测序（NGS）。为优化这种唾液DNA提取协议的目的是最大限度地提高数量和质量的遗传学应用，很容易与普通试剂和耗材实验室实现了一个具有成本效益的方式。 从唾液DNA提取需要几个步骤:1）收集并存储，2）细胞裂解，3）核糖核酸酶处理，4）沉淀蛋白，5）用乙醇沉淀，6）DNA的补液。该DNA的稳定缓冲液，所述的此前2，没有充分改变功能。没有尝试优化核糖核酸酶处理和DNA补液措施作出。对于每一个剩下的步骤，可能影响产量的几个变量进行鉴定。每个变量都被单独操纵和提高产量和质量统计评估。对于被证实可以改善产率和/或DNA质量的变量的最优值被包括在最终的协议。

<table border="0" cellpadding="0" cellspacing="0" width="260">
	<tbody>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">15ml Centrifuge Tubes</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">12-565-268</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Cell Lysis Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158908</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Proteinase K</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:84px;">P6556</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Protein Precipitation Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158912</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Isopropanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">A416-4</td>
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		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Glycogen</td>
			<td style="width:88px;">EZ-BioResearch</td>
			<td style="width:84px;">S1003</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">70% Ethanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">04-355-305</td>
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		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Tris-EDTA (TE)</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP2473-1</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">NaCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">AC194090010</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Tris HCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP1757-100</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">EDTA(0.5M) Solution</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">03-500-506</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Sodium Dodecyl Sulfate</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP166-100&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
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			<td></td>
			<td></td>
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			<td></td>
			<td></td>
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			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Name of Equipment</td>
			<td style="width:88px;">Distributor</td>
			<td style="width:84px;">Catalog#</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:88px;">Analog Vortex Mixer</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">02-215-365</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Centrifuge 5810R</td>
			<td style="width:88px;">Eppendorf</td>
			<td style="width:84px;">5811 000.010</td>
		</tr>
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</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.

为了确定最佳参数提取DNA进行了一系列配对的DNA提取的。单唾液样品被分裂并与两个可能的值对于给定的变量1的各部分进行测试。至少8个重复的每个配对测试被执行（ 例如 ，一个单唾液样品等分，以测试提取具有和不具有初始50℃温育）。优化是基于四个标准指标:总DNA的收量，二百八十零分之二百六十〇值时，二百三十分之二百六十值，和电泳的DNA以评估碎片的目视检查。变量不是所有...

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.

收集和唾液DNA提取用于新一代测序

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 de Cartagena UDEC

Research

JoVE Journal

Medicine

39.0K 次观看.  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.

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

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

测试:α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

生物标志物的研究下一代组织微阵列（ngTMA）协议

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

腹腔镜技术在猕猴肝和肠系膜淋巴结的串行采集

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.
Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing.&#160;Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.
In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

念珠菌光滑全基因组测序检测抗真菌耐药性标志物

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke survivors. The diagnosis and characterization of these symptoms in individual patients can be challenging and often requires skilled clinical staff. Virtual reality (VR) presents an opportunity to develop novel assessment tools for patients with USN.
We aimed to design and build a VR tool to detect and characterize subtle USN symptoms, and to test the tool on subjects treated with inhibitory repetitive transcranial magnetic stimulation (TMS) of cortical regions associated with USN.
We created three experimental conditions by applying TMS to two distinct regions of cortex associated with visuospatial processing- the superior temporal gyrus (STG) and the supramarginal gyrus (SMG) - and applied sham TMS as a control. We then placed subjects in a virtual reality environment in which they were asked to identify the flowers with lateral asymmetries of flowers distributed across bushes in both hemispaces, with dynamic difficulty adjustment based on each subject's performance. 
We found significant differences in average head yaw between subjects stimulated at the STG and those stimulated at the SMG and marginally significant effects in the average visual axis.
VR technology is becoming more accessible, affordable, and robust, presenting an exciting opportunity to create useful and novel game-like tools. In conjunction with TMS, these tools could be used to study specific, isolated, artificial neurological deficits in healthy subjects, informing the creation of VR-based diagnostic tools for patients with deficits due to acquired brain injury. This study is the first to our knowledge in which artificially generated USN symptoms have been evaluated with a VR task.

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

用于评估单边空间忽视的虚拟现实工具:数据收集的新机遇

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke ...