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).

참고 : 이전 타액 샘플을 제공하기 위해 모든 주제는 전국 어린이 병원에서 사람을 대상으로 치료를위한 가이드 라인을 준수 동의를 통보했다. 1 타액 수집 및 저장 <ol><li> 타액을 수집하기 전에 피사체의 입 피사체가 물로 입을 헹구어 갖는 식사를 피하거나 샘플을 수집하기 전에 30 분 동안 마시는 음식이나 이물질이 없는지 확인합니다. </li><li> 캡 또는 튜브의 내부에 손이 닿지 않도록 확인하는 DNA 안정화 버퍼 (2) 2.5 ㎖로 15 ㎖의 원심 분리기 튜브를 열고 피사체가 완충 용액에 침 2.5 ㎖의 침이있다. 참고 : 타액의 이상 2.5 ml의 수집은 DNA 안정화 버퍼에 샘플의 부족 비율에서 샘플이 저하 될 수 있습니다. 너무 작은 타액을 수집하는 프로토콜에서 예상 수확량을 감소시킬 것이다. 컬렉션 볼륨을 평가하려면, t 측에 번호 그라디언트를 사용그는 튜브. </li><li> 캡을 교체하고, 혼합물을 균질화 될 때까지 반전으로 섞는다. 격렬한 흔들림이 필요하지 않습니다. 단기 기억 또는 장기 저장을위한 4 ° C (&gt; 3 개월)에 대한 RT에서 샘플을 보관합니다. </li></ol> 2 초기 준비 및 세포 용해 <ol><li> 이전 추출을 시작으로, 37 ° C에 수조를 가열하고, 얼음 양동이를 준비합니다. 세 15 ML 원뿔 원심 분리기 튜브는 각 추출 된 샘플에 대해 필요합니다. 세 튜브 세포 단백질 펠렛, 최종 gDNA를 추출하고, 이소프로판올 및 에탄올 상청액을 보유하는 데 사용된다. </li><li> 15 초 후 중간 속도로 소용돌이를 저장에서 샘플 및 반전 샘플을 여러 번 검색합니다. </li><li> 깨끗한 15 ㎖의 원심 분리 관에 시료를 2.5 ㎖에 분배하고, 세포 용해 용액 5 ㎖를 추가합니다. 반전에 의해 시료 50 시간 혼합하고, 30 분 동안 RT에서 배양한다. </li></ol> 3 RNA 제거 <ol><li> 의 RNase 솔의 40 μl를 추가100 ㎎ / ㎖에서의 ution, 그리고 15 분 동안 37 ° C에서 배양한다. </li><li> 3 분 동안 얼음에 37 ° C의 물을 욕조에서 샘플을 제거하고 멋진. <ol><li> 의 RNase 인큐베이션 후, 프로토콜의 DNA의 재수 단계에서 65 ° C로 수조의 온도를 증가시킨다. </li></ol></li></ol> (4) 단백질 및 지질 제거 <ol><li> , ㎖ / 20 밀리그램에 테 K 솔루션의 50 μl를 추가 반전에 의해 여러 번 혼합과 30 분의 최소 RT 부화. 참고 :이 프로토콜에 대한 가능한 일시 정지 포인트입니다. 추출이 완료 될 때까지 K 테 수용액을 첨가 한 후, 샘플을 4 ° C에서 저장 될 수있다. 최대 24 시간 동안 4 ℃에서 보관 추출 수율 또는 DNA의 품질에 상당한 영향을 미치는 것으로 도시되지 않았다. 이 단계에서 장기 저장이 평가되지 않았습니다. </li><li> 10 분 동안 얼음에 20 고속 초, 및 장소를 적극적 소용돌이 단백질 침전 솔루션의 1.7 ML을 추가합니다. </l난&gt; <li> 샘플은 3000 XG에 10 분, 4 ° C에 10 분, 원심 분리기에 얼음에 냉각 한 후에. 침전 된 단백질은 단단한 펠릿이 계속 형성해야합니다. 펠릿을 꽉 없거나 흐린 용액이 여전히 있다면, 샘플은 5 분 더 얼음 위에서 냉각 할 수 있고, 원심 분리를 반복. 샘플은 단단한 펠릿을 보장하기 위해 얼음에 보관해야합니다. </li></ol> (5) 분리 및 gDNA를 정제 <ol><li> 깨끗한 15 ㎖의 원심 분리 관에, 피펫 아이소 프로필 알코올 5 ㎖ 및 20 ㎎ / ㎖에서 순수 글리코겐 솔루션의 8 μL. </li><li> 침전 된 단백질 펠렛을 남겨두고, 아이소 프로필 알코올과 글리코겐 솔루션을 포함하는 튜브에 4.3 단계에서 gDNA를가 포함 된 상층 액을 붓는다. 상층 액을 추가 한 후, 부드럽게 3000 XG 및 4 ℃에서 30 분 동안 샘플을보기 반전 원심 분리에 의해 50 시간 혼합한다. </li><li> 깨끗한 15 ML 튜브에 천천히 뜨는을 붓고. 상청액을 제거한 후, 70 % 에탄올 1 ㎖를 추가 할수회 천천히 부드럽게 흔들 석출 펠렛 위에 에탄올을 이동하여 펠렛을 씻는다. 튜브에서 에탄올을 유지합니다. </li><li> 초기 세척 한 후, 2000 XG에 1 분, 20 ° C에 대한 샘플을 원심 분리기. 이 원심 분리 단계는 4 ° C에서 20 ° C 중 하나에서 수행 할 수 있습니다. 온도의 큰 영향이 단계에서는 도시되지 않았다. </li><li> 펠렛의 초기 세척 및 원심 분리 후, 천천히 튜브에서 에탄올 세척을 부어 버리고, 다음 단계 5.3 및 5.4를 반복하여 두 번째 세척을 수행합니다. </li><li> 두 번째 세척에서 상층 액의 제거 후, ​​15 분간 자연 건조 펠렛을 할 수 있습니다. <ol><li> 시료를 완전히 건조되지 않은 경우, 또 다른 15 분 동안 공기 건조. </li></ol></li></ol> gDNA를의 6 재수 <ol><li> 샘플이 건조되면, 건조 gDNA를 펠렛을 재수하는 트리스 - EDTA (에틸렌 다이아 민 테트라 초산) 300 μl를 추가합니다. </li><li> 중간 속도와 장소에서 5 초 동안 소용돌이 샘플1 시간 동안 65 ° C 온수 목욕입니다. </li><li> 수조에서 샘플을 제거하고 RT에서 O / N을 품어. 참고 : 모든 제품 및 시약 재료 표에 나와있는 사용뿐만 아니라, 표 4. </li></ol>

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The authors have nothing to disclose.

본 절차는 상당히 DNA의 품질을 손상시키지 않고, 표준 방법에 비해 고 분자량 DNA의 수율이 향상되었다 최적화 DNA 추출 프로토콜이다. 수율을 최대한에 가장 큰 효과 중요한 단계 널리 11을 배포하지 않은 일을 제외하고, 여기에서 검토 게시 된 프로토콜에 비해 에탄올 침전 동안 이상 원심 분리 단계를 포함하는 단계 5.2였다. 이 이상 원심 분리와 관련된 DNA 품질의 변경은 전체 타액 컬렉션...

인간 유전 연구 고품질 DNA를 얻는 것은 질병 유전자 발견 프로세스에 필수적이다. 혈액, 타액 수집보다 더 비싼 또한 침략적인 절차를 요구하고 있지만, 기능 연구에 대한 무한 DNA의 소스 또는 iPSCs로 불후의 세포 라인을 만드는 선호하고, 세포주를 사용할 수없는 경우 때때로 혈액 DNA가 사용됩니다. 그러나, 혈액을 획득하는 훈련 된 phlebotomist을 필요로 피가 침 하나보다 짧은 반감기를 가지고있다. 그것을 수집함으로써 잘 병원 실험실 (2)의 집수 영역을 넘어 전위 피사체 풀을 증가 phlebotomist 필요없이 이메일을 통해 전송 될 수 있기 때문에 타액으로부터 DNA는 얻을 저렴하고 용이하다. 과목 대신 혈액 3, 4의 타액 샘플을 채취 할 수있는 옵션이있을 때 연구 등록이 개선 될 수있다. 수량과 타액에서 DNA의 질에 대한 우려는 우리가 널리 제한되어 있습니다타액 2, 3, 4, 5의 상당한 양을 구하는 않았다 이전 협측 면봉 방법을 통해 DNA 테스트에 대한 밀리리터 당 4.3 × 105 세포의 평균으로, 전체 타액의 적합성을 나타내는 수많은 연구 최근의 연구에도 불구하고, 즉, 6. 겸손한 문학 마이크로 어레이 기반 방식 팔을 포함하여 유전자형 응용 프로그램에 대한 전체 타액 유래 DNA의 적합성을 보여주는 존재하지만 9, 10, 더 연구가 시도되지 않은 차세대 시퀀싱 (NGS). 이 모든 타액 DNA 추출 프로토콜을 최적화하는 목적은 쉽게 일반적인 시약 및 소모품으로 실험실에서 구현 비용 효과적인 방식으로 응용 프로그램에 대한 유전학 수량 및 품질을 최대화하는 것이다. 타액에서 DNA 추출은 여러 절차가 필요합니다 : 1) 수집 및 저장, 2) 세포 용해, 3)의 RNase 처리, 4) 단백질 침전, 5) 에탄올 침전, 6) DNA의 수분 보충을. 기재된 DNA 안정화 버퍼 용액이전에이 적절하게 변경없이 작동합니다. 의 RNase 처리 및 DNA의 수분 보충 단계를 최적화하기위한 어떠한 시도도하지 않았다. 나머지 각 단계의 수율에 영향을 미칠 수있는 여러 변수가 확인되었다. 각 변수를 개별적으로 조작하고, 수율 및 품질의 향상이 통계적으로 평가 하였다. 수율 및 / 또는 DNA의 품질을 향상시키기 위해 도시 된 변수의 최적 값은 최종 프로토콜에 포함되었다.

<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.

DNA는 한 쌍의 DNA를 추출하는 일련의이 수행 된 추출을위한 최적의 매개 변수를 결정합니다. 하나의 타액 샘플을 분할하고 각 부분은 주어진 변수에 대한 두 가지 값 중 하나를 사용하여 테스트되었습니다. 각 쌍 테스트 적어도 8 회 반복 (예를 들어, 하나의 타액 샘플과 초기 50 ° C에서 배양없이 모두 추출을 테스트하기 위해 분주했다)을 시행 하였다. 총 DNA 수율, 280 분의 260 값 230분의 260 값...

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.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

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 조회수.  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.

비디오: 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.

목초의 군 유전체학 분석

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

연구

유전학

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.

자동 젤 사이즈 선택은 환경 물 샘플에서 준비 차세대 시퀀싱 라이브러리의 질을 향상시키기 위해

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

환경과학

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.

차세대 시퀀싱에 적합한 식물성 세포질 DNA 농축 방법의 최적화 및 비교 분석

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.

롤리팝 - 같은 Ultrafiltration 프로브를 사용하여 인간의 원주민 타액 Peptidome를 샘플링 : 임상 질량 분광법을위한 펩타이드 감지를 단순화하고 향상

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

의학

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.

실험실 클래스에서 D1S80 로커스를 사용하여 DNA 지문 응용 프로그램

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.

높은 처리량의 사용<em&gt; 체외</em&gt; 미세 유체 시스템 구강 다중 종의 바이오 필름을 개발하는

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

생체공학

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.

짧고 긴 읽기 시퀀싱 기술을 사용하여 요로나박테리아의 완전한 게놈 생성을 위한 하이브리드 드 노보 게놈 조립

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.

새로운 바이러스 게놈을 발견 감염 된 잎에서 RNA 분석 원유 Virion 추출에 DNA의 결합 분석

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

면역학 및 감염병학

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.

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

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