우리는 테일러 AuBuchon-엘 더, 요르단 Teisher, 및 샘플링 표 본관 표본에 크리스티나 Zudock와 미주리 식물원 표 본관 표본에 파괴적인 샘플링에 대 한 액세스에 대 한 감사합니다. 이 작품은 국립 과학 재단 (뎁-1457748)에서 교부 금에 의해 지원 이었다.

<ol>
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저자 들은 아무 경쟁 관심사를 선언 합니다.

여기에 제시 된 프로토콜 DNA 고립과 말린된 식물 견본에서 라이브러리 준비 시퀀싱에 대 한 포괄적이 고 강력한 방법입니다. 방법과 그것을 변경 하는 최소한의 필요가 일관성 기반 견본 품질에 큰 표 본관 기반 시퀀싱 프로젝트에 대 한 확장 가능한 그것. 낮은 수익률 라이브러리에 대 한 선택적 reamplification 단계 포함을 낮은 품질, 낮은 수량, 드문, 또는 시퀀싱에 적합 되지 않을 역사적으로 중요...

표 본관 컬렉션은 종 및 계통학1,2,3, 인구 유전학4,5, 보존을 포함 하 여 연구에 대 한 게놈 다양성의 잠재적으로 귀중 한 소스 생물학6, 침략 적인 종의 생물학7, 그리고 특성 진화8. 다양 한 종, 인구, 지리적 위치, 및 시간 포인트를 얻을 수 있는 식물 표본 상자 "보물 상자"9 를 강조 표시 합니다. 역사적으로, 표 본관 파생 DNA의 저하 자연 PCR 기반 프로젝트, 종종 높은 복사, 엽록체 게놈 또는 ribosomal의 내부 베낀된 스페이서 (ITS)의 지역 등에 마커를 사용 하 여 연구자를 일하고 방해 했다 RNA입니다. 표본 및 DNA의 품질 보존9,10, 더블-좌초 나누기와 손상의 가장 흔한 형태 되 고, 건조 과정에서 사용 되는 열에서 조각화의 방법에 광범위 하 게 따라 다는 소위 90 %DNA 잠금 업 된 PCR 기반 연구11지 장 있다. 조각화, 외 표 본관 유전체학에서 두 번째로 가장 널리 퍼진 문제는 오염, 그13 endophytic 균 류 파생 된 또는 수집 후 하지만 그래도 표 본관12에 장착 하기 전에 곰 팡이 사후 인수 이 문제 해결된 bioinformatically 바로 곰 팡이 데이터베이스 (아래 참조) 제공 될 수 있습니다. 세 번째, 및 보다 적게 일반적인 문제는 비록 표 본관 표본11(~ 0.03%) 낮은 것으로 추정 된다 시 토 신 탈 (C/G→T/A)14, 통해 시퀀스 수정 이다. 높은 처리량 시퀀싱 (HTS)의 출현, 조각의 문제는 짧은 읽기 및 시퀀싱 깊이12,15, 낮은 질을 가진 수많은 표본에서 게놈 수준의 데이터 수집을 허용으로 극복할 수 있습니다. DNA, 그리고 때로는 심지어 전체 게놈 시퀀싱15허용.
식물 표본 상자 견본 더 자주 사용 되 고 있으며 계통 발생 프로젝트16의 큰 구성 요소. HTS에 대 한 식물 표본 상자 견본을 사용 하 여 현재 도전 개인에 대 한 메서드를 최적화 하지 않고도 충분 한 더블 좌초 DNA 시퀀싱 프로토콜에 대 한 필요한 필수 적절 한 시기에 수많은 종에서 얻는 지속적으로 표본입니다. 이 논문에서는, DNA 추출 및 표 본관 표본 라이브러리 준비에 대 한 프로토콜을 기존의 방법의 활용 및 결과 신속 하 고 복제할 수 있도록 그들을 수정 시연입니다. 이 방법은 완전 한 13 h 8 h 실습 시간, 또는 9 h 실습 시간 때 옵션 reamplification 단계는 필요와 함께 16 h 24 샘플 라이브러리에 견본에서 처리 수 있습니다. 더 많은 샘플의 동시 처리는 달성, 비록 제한 요소는 원심 분리기 용량 및 기술. 프로토콜은 DNA를 전단에 대 한만 전형적인 실험실 장비 (thermocycler, 원심 분리기, 마그네틱 스탠드) 대신 분무기 또는 sonicator, 특수 장비를 요구 하도록 설계 되었습니다.
DNA 품질, 조각 크기 및 수량 표 본관 표본 높은 처리량 시퀀싱 실험에서의 사용에 대 한 요인 제한. 식물 표본 상자 DNA를 분리 하 고 높은 처리량 시퀀싱 라이브러리를 만들기 위한 다른 방법을 10 만큼 약간을 사용 하 여 유틸리티를 증명 하고있다 ng의 DNA16; 그러나 라이브러리 준비에 필요한 사이클 그들은 실험적으로 PCR의 최적 수를 결정 해야 합니다. 이것은 된다 허무 다루는 상당히 적은 양의 가능한 더블 좌초 DNA (dsDNA) 때 일부 표 본관 표본 단일 라이브러리 준비에 대 한 충분 한 DNA를 생산. 여기에 제시 된 방법 아무 DNA 라이브러리 최적화 단계에서 분실 된다 그래서 샘플 품질에 사이클의 단일 번호를 사용 합니다. 대신, reamplification 단계 라이브러리 시퀀싱에 필요한 최소 금액을 충족 하지 않는 경우 호출 됩니다. 많은 식물 표본 상자 견본 드물다 고 많은 경우에 파괴적인 샘플링을 정당화 하기 어려운 작은 재료를가지고. 이 카운터, 제시 프로토콜 허용 dsDNA 입력 크기 미만 1.25 ng 도서관 준비 과정, 높은 처리량 시퀀싱 및 표본의 파괴적인 샘플링에 대 한 필요를 최소화에 대 한 실행 가능한 샘플의 범위를 확대.
다음 프로토콜 잔디에 최적화 되었고 비록 우리가 기대 하는 다른 많은 식물 그룹에 프로토콜을 적용할 수 있는 식물 표본 상자 견본에서 다른 종의 수백에 테스트. 그것은 낮은 품질 및 희귀 표본 저장 하는 데 사용할 수 있는 선택적 복구 단계를 포함 합니다. 이상 2 백 표 본관 표본 테스트에 따라,이 프로토콜 표본 낮은 조직 입력 및 품질, 최소한의 파괴적인 샘플링을 통해 희귀 한 표본의 보존에 대 한 허용에 작동 합니다. 여기이 프로토콜 phylogenomics 기반 프로젝트에 대 한 시퀀싱 할 수 있는 높은 품질 라이브러리를 제공할 수 있습니다 표시 됩니다.

<table><tbody><tr><td>Veriti Thermal Cycler</td><td>Applied Biosystems</td><td>4452300</td><td>96 well&amp;nbsp;</td></tr><tr><td>Gel Imaging System</td><td>Azure Biosystems</td><td>c300</td><td></td></tr><tr><td>Microfuge 20 Series</td><td>Beckman Coulter</td><td>B30137</td><td></td></tr><tr><td>Digital Dry Bath</td><td>Benchmark Scientific</td><td>BSH1001</td><td></td></tr><tr><td>Electrophoresis System</td><td>EasyCast</td><td>B2</td><td></td></tr><tr><td>PURELAB flex 2 (Ultra pure water)</td><td>ELGA&amp;nbsp;</td><td>89204-092</td><td></td></tr><tr><td>DNA LoBind Tube&amp;nbsp;</td><td>Eppendorf</td><td>30108078</td><td>2 ml</td></tr><tr><td>Mini centrifuge</td><td>Fisher Scientific</td><td>12-006-901</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Mortar</td><td>Fisher Scientific</td><td>S02591</td><td>porcelain</td></tr><tr><td>Pestle</td><td>fisher Scientific</td><td>S02595</td><td>porcelain</td></tr><tr><td>Centrifuge tubes</td><td>fisher Scientific</td><td>21-403-161</td><td></td></tr><tr><td>Microwave</td><td>Kenmore</td><td>405.7309231</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>0.2 ml Strip tube and Cap for PCR</td><td>VWR</td><td>20170-004</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>PM2000</td><td></td></tr><tr><td>Liquid Nitrogen Short-term Storage</td><td>Nalgene</td><td>F9401</td><td></td></tr><tr><td>Magnetic-Ring Stand&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>AM10050</td><td>96 well&amp;nbsp;</td></tr><tr><td>Water Bath</td><td>VWR</td><td>89032-210</td><td></td></tr><tr><td>Hot Plate Stirrers</td><td>VWR</td><td>97042-754</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Airgas</td><td>UN1977</td><td></td></tr><tr><td>1 X TE Buffer</td><td>Ambion</td><td>AM9849</td><td>pH 8.0</td></tr><tr><td>CTAB</td><td>AMRESCO</td><td>0833-500G</td><td></td></tr><tr><td>2-MERCAPTOETHANOL</td><td>AMRESCO</td><td>0482-200ML</td><td></td></tr><tr><td>Ribonuclease A</td><td>AMRESCO</td><td>E866-5ML</td><td>10 mg/ml solution</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63882</td><td></td></tr><tr><td>Sodium Chloride</td><td>bio WORLD</td><td>705744</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>bio WORLD</td><td>40970004-1</td><td></td></tr><tr><td>Nuclease Free water</td><td>bio WORLD</td><td>42300012-2</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Fisher Scientific</td><td>A393-500</td><td></td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Fisher Scientific</td><td>s608-500</td><td></td></tr><tr><td>LE Agarose</td><td>GeneMate</td><td>E-3120-500</td><td></td></tr><tr><td>100bp PLUS DNA Ladder</td><td>Gold Biotechnology</td><td>D003-500</td><td></td></tr><tr><td>EDTA, Disodium Salt</td><td>IBI Scientific</td><td>IB70182</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Life Technologies</td><td>Q32854</td><td></td></tr><tr><td>TRIS</td><td>MP Biomedicals</td><td>103133</td><td>ultra pure</td></tr><tr><td>Gel Loading Dye Purple (6 X)</td><td>New England BioLabs</td><td>B7024S</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England BioLabs</td><td>M0348L</td><td></td></tr><tr><td>NEBNext Ultra II DNA Library Prep Kit for Illumina&amp;nbsp;</td><td>New England BioLabs</td><td>E7645L</td><td></td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>New England BioLabs</td><td>E7600S</td><td>Dual Index Primers Set 1</td></tr><tr><td>NEBNext Q5 Hot Start HiFi PCR Master Mix</td><td>New England BioLabs</td><td>M0543L</td><td></td></tr><tr><td>Mag-Bind RXNPure Plus</td><td>Omega bio-tek</td><td>M1386-02</td><td></td></tr><tr><td>GelRed 10000 X</td><td>Pheonix Research</td><td>41003-1</td><td></td></tr><tr><td>Phenol solution</td><td>SIGMA Life Science</td><td>P4557-400ml</td><td></td></tr><tr><td>PVP40</td><td>SIGMA-Aldrich</td><td>PVP40-50G</td><td></td></tr><tr><td>Chloroform</td><td>VWR</td><td>EM8.22265.2500</td><td></td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1016</td><td>200 Proof</td></tr><tr><td>Silica sand</td><td>VWR</td><td>14808-60-7</td><td></td></tr><tr><td>Reamplification primers</td><td>Integrated DNA Technologies</td><td>see text</td><td></td></tr><tr><td>Sequencher v.5.0.1</td><td>GeneCodes</td><td></td><td></td></tr></tbody></table>

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

Herbaria는 다양 한 생물 학적 연구에에서 사용할 수 있는 공장 설비 재료의 귀중 한 소스. 표 본관 표본을 사용 하 여 샘플 보존 품질, 성능이 저하 된 DNA 및 희귀 표본의 파괴적인 샘플링을 포함 하 여 과제의 번호와 연결 됩니다. 큰 시퀀싱 프로젝트에서 식물 표본 상자 물자를 더 효과적으로 사용 하려면 DNA 고립과 라이브러리 준비는 신뢰할만 하 고 확장 가능한 방법 필요 합니다. 이 종이 DNA 분리 및 높은 처리량 도서관 건설 표 본관 표본에서 개별 샘플에 대 한 수정 요구 하지 않는 대 한 강력 하 고, 시작-투-엔드 프로토콜을 보여 줍니다. 이 프로토콜은 기존 방법의 낮은 질 건조 식물 재료와 소요 장점에 대 한 최적화, 조직 분쇄 라이브러리 크기 선택, 수정 하 고 낮은 수율 라이브러리에 대 한 선택적 reamplification 단계를 도입 하 여 지어진 다. 낮은 수익률 DNA 라이브러리의 reamplification 샘플 인지할 시퀀싱 바이어스 공통에 대 한 소개 없이 추가 파괴적인 샘플링에 대 한 필요성을 negating 대신할 및 잠재적으로 귀중 한 식물 표본 상자 견본에서 파생 된 구조 수 있습니다. 계통 발생 응용 프로그램입니다. 프로토콜 잔디 종, 수백에서 테스트 되었습니다 하지만 확인 후 다른 식물 계보에 사용 하기 위해 적응 될 것으로 예상 된다. 이 프로토콜 어디 조각 원하는 크기로 범위에 존재 하지 않는, 매우 저하 DNA와 보조 metabolites 일부 공장 설비 재료에 깨끗 한 DNA 분리를 억제 하는 제한 될 수 있습니다. 전반적으로,이 프로토콜 DNA 분리 및 활성 실습 시간 최소한의 수정만 8 h 13 h 24 샘플 라이브러리 준비 수 신속 하 고 포괄적인 방법을 소개 합니다.

DNA 분리 및 최종 라이브러리 수율 본이 연구에서는 표 본관 DNA의 고립과 고품질 시퀀싱 라이브러리의 복구에 대 한 프로토콜의 효능은 50 다른 샘플을 사용 하 여 1920 년에서 가장 오래 된와 2012 (표 2)에서 최 연소 시연 했다. 각 샘플에 대 한 약 10 mg의 잎 조직 DNA 격리 사용 되었다. 가능한 경우, 녹색 잎 조직이 선호 했다 그리고 확실 한 곰 팡이 ?...

Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

강력한 DNA 고립과 표 본관 표본에 대 한 높은 처리량 시퀀싱 도서관 건축

이 문서에서는 DNA 분리 및 매우 가난한 품질 DNA의 구조를 포함 하 여 식물 표본 상자 물자에서 높은 처리량 시퀀싱 도서관 건축을 위한 상세한 프로토콜을 보여 줍니다.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

robust-dna-isolation-high-throughput-sequencing-library-construction

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JoVE Journal

Genetics

10.5K 조회수.  Donald Danforth Plant Science Center. 이 문서에서는 DNA 분리 및 매우 가난한 품질 DNA의 구조를 포함 하 여 식물 표본 상자 물자에서 높은 처리량 시퀀싱 도서관 건축을 위한 상세한 프로토콜을 보여 줍니다.

비디오: Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

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 ...

연구

환경과학

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

긴 읽기 시퀀싱에 대한 흰가루병에서 높은 분자량의 게놈의 DNA의 정제

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

유전학

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 ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

실험, Metagenomic, 및 계산 기법 밝히는 어떤 균 타협 꿀벌 건강 메커니즘

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Ultralow 입력된 게놈 시퀀싱 단일 Tardigrade 견본에서 라이브러리 준비

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

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 ...

<meta charset="utf8"></head><body>약용 식물을 위한 DNA 바코드: 식별 및 계통 발생 분석

Application of DNA Barcoding to Identify Medicinal Plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

<meta charset="utf8"></head><body>저자 스포트라이트: DNA 바코드 기술을 활용하여 약용 식물 식별의 효율성 향상

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

약용 식물을 식별하기 위한 DNA 바코드 적용

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

젤-seq: DNA와 RNA 히드로 매트릭스를 사용 하 여 동시 시퀀싱 라이브러리 준비를 위한 방법

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

강력한 DNA 고립과 표 본관 표본에 대 한 높은 처리량 시퀀싱 도서관 건축

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

긴 읽기 시퀀싱에 대한 흰가루병에서 높은 분자량의 게놈의 DNA의 정제

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

실험, Metagenomic, 및 계산 기법 밝히는 어떤 균 타협 꿀벌 건강 메커니즘

Ultralow 입력된 게놈 시퀀싱 단일 Tardigrade 견본에서 라이브러리 준비

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

약용 식물을 식별하기 위한 DNA 바코드 적용

약용 식물을 식별하기 위한 DNA 바코드 적용

약용 식물을 식별하기 위한 DNA 바코드 적용

젤-seq: DNA와 RNA 히드로 매트릭스를 사용 하 여 동시 시퀀싱 라이브러리 준비를 위한 방법