Name: optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing
Uploaded: 2017-07-28T12:30:00.000+00:00
Duration: 12 min 33 s
Description: Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

우리는 미국 농무부 농업 연구청과 국립 과학 재단 (IOS 1025881 및 IOS 1361554)으로부터 자금을 지원하고 싶습니다. 우리는 R. Caspers에게 온실 유지 관리 및 식물 관리에 감사드립니다. 우리는 Illumina 라이브러리 준비 및 시퀀싱이 수행 된 Minnesota Genomics Center 대학에도 감사드립니다. 저널 편집자와 4 명의 익명의 평론가가 우리 원고를 더욱 강화시킨 것에 대해 감사드립니다. 우리는 또한 OECD가 일본의 동료들과 협력 프로젝트를 위해 이러한 프로토콜을 통합하는 SK와의 협력에 감사한다.

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	<li>Liberatore, K. L., Dukowic-Schulze, S., Miller, M. E., Chen, C., Kianian, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+plant+development+and+stress+tolerance.">The role of mitochondria in plant development and stress tolerance.</a> Free Radic Biol Med. 100, 238-256 (2016).</li><li>Samaniego Castruita, J. A., Zepeda Mendoza, M. L., Barnett, R., Wales, N., Gilbert, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odintifier--A+computational+method+for+identifying+insertions+of+organellar+origin+from+modern+and+ancient+high-throughput+sequencing+data+based+on+haplotype+phasing.">Odintifier--A computational method for identifying insertions of organellar origin from modern and ancient high-throughput sequencing data based on haplotype phasing.</a> BMC Bioinformatics. 16 (232), 1-13 (2015).</li><li>Zhang, T., Zhang, X., Hu, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+procedure+for+plant+organellar+genome+assembly,+based+on+whole+genome+data+from+the+454+GS+FLX+sequencing+platform.">An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform.</a> Plant Methods. 7 (38), 1-8 (2011).</li><li>Wambugu, P. W., Brozynska, M., Furtado, A., Waters, D. L., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+wild+and+domesticated+rices+(Oryza+AA+genome+species)+based+upon+whole+chloroplast+genome+sequences.">Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences.</a> Sci Rep. 5 (13957), 1-9 (2015).</li><li>Iorizzo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+assembly+of+the+carrot+mitochondrial+genome+using+next+generation+sequencing+of+whole+genomic+DNA+provides+first+evidence+of+DNA+transfer+into+an+angiosperm+plastid+genome.">De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome.</a> BMC Plant Biol. 12 (61), 1-17 (2012).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+sequences+of+organelle+genomes+from+the+medicinal+plant+Rhazya+stricta+(Apocynaceae)+and+contrasting+patterns+of+mitochondrial+genome+evolution+across+asterids.">Complete sequences of organelle genomes from the medicinal plant Rhazya stricta (Apocynaceae) and contrasting patterns of mitochondrial genome evolution across asterids.</a> BMC Genomics. 15 (405), 1-18 (2014).</li><li>Skippington, E., Barkman, T. J., Rice, D. W., Palmer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+mitogenome+of+the+parasitic+plant+Viscum+scurruloideum+is+extremely+divergent+and+dynamic+and+has+lost+all+nad+genes.">Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes.</a> Proc Natl Acad Sci U S A. 112 (27), E3515-E3524 (2015).</li><li>Wicke, S., Schneeweiss, G. M., H&#246;randl, E., Appelhans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.">Chapter 1.</a> Next Generation Sequencing in Plant Systematics. , (2015).</li><li>Sloan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+ring+to+rule+them+all?+Genome+sequencing+provides+new+insights+into+the+'master+circle'+model+of+plant+mitochondrial+DNA+structure.">One ring to rule them all? Genome sequencing provides new insights into the 'master circle' model of plant mitochondrial DNA structure.</a> New Phytol. 200 (4), 978-985 (2013).</li><li>Woloszynska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteroplasmy+and+stoichiometric+complexity+of+plant+mitochondrial+genomes--though+this+be+madness,+yet+there's+method+in't.">Heteroplasmy and stoichiometric complexity of plant mitochondrial genomes--though this be madness, yet there's method in't.</a> J Exp Bot. 61 (3), 657-671 (2010).</li><li>Ahmed, Z., Fu, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+with+a+wider+applicability+to+isolate+plant+mitochondria+for+mtDNA+extraction.">An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction.</a> Plant Methods. 11 (56), 1-11 (2015).</li><li>Ejaz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+small+scale+methods+for+the+rapid+and+efficient+extraction+of+mitochondrial+DNA+from+wheat+crop+suitable+for+down-stream+processes.">Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes.</a> Genet Mol Res. 13 (4), 10320-10331 (2014).</li><li>Eubel, H., Heazlewood, J. L., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+plant+mitochondria+for+proteomic+analysis.">Isolation and subfractionation of plant mitochondria for proteomic analysis.</a> Methods Mol Biol. 355, 49-62 (2007).</li><li>Hao, W., Fan, S., Hua, W., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+extraction+and+assembly+methods+for+simultaneously+obtaining+plastid+and+mitochondrial+genomes.">Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.</a> PLoS One. 9 (9), e108291 (2014).</li><li>Pomeroy, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+respiratory+properties+of+mitochondria+isolated+from+developing+winter+wheat+seedlings.">Studies on the respiratory properties of mitochondria isolated from developing winter wheat seedlings.</a> Plant Physiol. 53 (4), 653-657 (1974).</li><li>Taylor, N. L., Stroher, E., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+organelle+isolation+and+characterization.">Arabidopsis organelle isolation and characterization.</a> Methods Mol Biol. 1062, 551-572 (2014).</li><li>Triboush, S. O., Danilenko, N. G., Davydenko, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+isolation+of+chloroplast+DNA+and+mitochondrial+DNA+from+Sunflower.">A method for isolation of chloroplast DNA and mitochondrial DNA from Sunflower.</a> Plant Mol Biol Rep. 16 (2), 183-189 (1998).</li><li>Pinard, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+whole+genome+amplification-induced+bias+through+high-throughput,+massively+parallel+whole+genome+sequencing.">Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.</a> BMC Genomics. 7 (216), 1-21 (2006).</li><li>Lamble, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+workflows+for+high+throughput+library+preparation+using+the+transposome-based+Nextera+system.">Improved workflows for high throughput library preparation using the transposome-based Nextera system.</a> BMC Biotechnol. 13 (104), 1-10 (2013).</li><li>Raley, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+next-generation+DNA+sequencing+libraries+from+ultra-low+amounts+of+input+DNA:+Application+to+single-molecule,+real-time+(SMRT)+sequencing+on+the+Pacific+Biosciences+RS+II.">Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.</a> bioRxiv. , (2014).</li><li>Tsai, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+Complexity+of+Human+Skin+Metagenomes+Using+Single-Molecule+Sequencing.">Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing.</a> MBio. 7 (1), e01948 (2016).</li><li>Feehery, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+selectively+enriching+microbial+DNA+from+contaminating+vertebrate+host+DNA.">A method for selectively enriching microbial DNA from contaminating vertebrate host DNA.</a> PLoS One. 8 (10), e76096 (2013).</li><li>Yigit, E., Hernandez, D. I., Trujillo, J. T., Dimalanta, E., Bailey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+and+metagenome+sequencing:+Using+the+human+methyl-binding+domain+to+partition+genomic+DNA+derived+from+plant+tissues.">Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues.</a> Appl Plant Sci. 2 (11), 1-6 (2014).</li><li>Noyszewski, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+evolution+of+the+mitochondrial+genome+in+an+alloplasmic+line+of+durum+wheat.">Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.</a> BMC Genomics. 15 (67), 1-16 (2014).</li><li>. QIAamp DNA Mini and Blood Mini Handbook Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2016)</li><li>. User developed protocol: Isolation of genomic DNA from plants and filamentous fungi using the QIAGEN Genomic-tip - (EN) Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2001)</li><li>. QIAGEN Genomic DNA Handbook Available from: <a target="_blank" href="https://www.qiagen.com/ch/resources/">https://www.qiagen.com/ch/resources/</a> (2012)</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Ogihara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+cereal+mitochondrial+genomes+as+revealed+by+complete+nucleotide+sequencing+of+the+wheat+mitochondrial+genome.">Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.</a> Nucleic Acids Res. 33 (19), 6235-6250 (2005).</li><li>Ogihara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+features+of+a+wheat+plastome+as+revealed+by+complete+sequencing+of+chloroplast+DNA.">Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.</a> Mol Genet Genomics. 266 (5), 740-746 (2002).</li><li>International Wheat Genome Sequencing Consortium (IWGSC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromosome-based+draft+sequence+of+the+hexaploid+bread+wheat+(Triticum+aestivum)+genome.">A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome.</a> Science. 345 (6194), (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Methods. 9 (4), 357-359 (2012).</li><li>Bendich, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+chloroplasts+and+mitochondria+contain+so+many+copies+of+their+genome?.">Why do chloroplasts and mitochondria contain so many copies of their genome?.</a> Bioessays. 6 (6), 279-282 (1987).</li><li>Kumar, R. A., Oldenburg, D. J., Bendich, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+DNA+damage,+molecular+integrity,+and+copy+number+for+plastid+DNA+and+mitochondrial+DNA+during+maize+development.">Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.</a> J Exp Bot. 65 (22), 6425-6439 (2014).</li><li>Ma, J., Li, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+genome+copy+number+variation+and+integrity+during+moderate+maturation+of+roots+and+leaves+of+maize+seedlings.">Organellar genome copy number variation and integrity during moderate maturation of roots and leaves of maize seedlings.</a> Curr Genet. 61 (4), 591-600 (2015).</li><li>Lang, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+isolation+of+pure+and+intact+chloroplasts+and+mitochondria+from+moss+as+the+basis+for+sub-cellular+proteomics.">Simultaneous isolation of pure and intact chloroplasts and mitochondria from moss as the basis for sub-cellular proteomics.</a> Plant Cell Rep. 30 (2), 205-215 (2011).</li><li>Tobin, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+of+plant+tissues.+Isolation+of+chloroplasts+and+mitochondria+from+leaves.">Subcellular fractionation of plant tissues. Isolation of chloroplasts and mitochondria from leaves.</a> Methods Mol Biol. 59, 57-68 (1996).</li></ol>

저자는 경쟁적 이익이 없다고 선언합니다. 이 간행물의 상호 또는 상업 제품에 대한 언급은 특정 정보를 제공하기위한 목적 일 뿐이며 미 농무부 (US Department of Agriculture)의 추천이나 보증을 의미하지는 않습니다. USDA는 기회 균등 제공자이자 고용주입니다.

지금까지 대부분의 organellar sequencing 연구는 특정 DC를 풍부하게하는 전통적인 DC 방법에 초점을두고 있습니다. 모스 ( 40)를 포함하여 다양한 식물로부터 세포 소기관을 분리하는 방법이 설명되었다. 밀 15 및 귀리 11 과 같은 단핵구; 아라비돕시스 11 , 해바라기 17 , 유채 14 등의 족자입니다. ?...

<html> 게놈 시퀀싱은 중요한 식물 형질의 기본 유전 적 기초를 분석하는 강력한 도구입니다. 대부분의 게놈 시퀀싱 연구는 대부분의 유전자가 핵에 위치하므로 핵 게놈 내용에 초점을 맞 춥니 다. 그러나, (진핵 생물에서) 미토콘드리아와 색소체 포함 organellar 게놈 (식물, 전문화 된 형태로, 엽록체, 광합성에서 작동)는 생명체의 개발, 스트레스 반응 및 전반적인 체력 1에 필수적인 중요한 유전 정보를 기여한다. Organellar 게놈은 일반적으로 핵 게놈 시퀀싱을위한 총 DNA 추출에 포함되지만 DNA 추출 이전에 세포 소기관 수를 줄이는 방법도 사용됩니다 2 . 많은 연구에서 전체 gDNA 추출물의 sequencing 결과를 사용하여 organellar 게놈 3 , 4 , 5 ,xref &quot;&gt; 6 , 7. 그러나 연구 대상이 세포 소기관 게놈에 초점을 맞추고있을 때 많은 gDNA가 핵산 염기 서열에&quot;손실 &quot;되어 있기 때문에 전체 핵산을 사용하면 시퀀싱 비용이 증가합니다. 적당한 게놈, bioinformatically 8 도전 2에 관한 것이다. 또한, 염기 서열의 정확한지도 위치를 해결 중복 핵 게놈 내로 소기관 사이 organellar 서열의 전사에 의한 읽기. 핵 게놈 organellar 게놈의 정제 하나이다 전략을 사용하여 미토콘드리아와 엽록체 사이의 상 동성 영역으로 매핑되는 판독을 분리 할 수 ​​있습니다. 많은 식물 종의 세포 소기관 게놈이 서열화되어 있지만, 소기관 게놈 다양성의 폭은 거의 알려져 있지 않습니다야생 개체군 또는 경작 된 육종 풀에서 이용 가능하다. Organellar 게놈은 또한 반복 서열 9 사이의 재조합으로 인해 중요한 구조적 재 배열을 거친 동적 분자로 알려져있다. 또한, 세포 소기관 게놈의 여러 사본이 각 세포 소기관에 포함되어 있으며, 여러 세포 소기관이 각 세포 내에 포함되어 있습니다. 이 게놈의 모든 사본이 동일하다는 것은 이질균이라고 알려져 있습니다. &quot;마스터 서클&quot;의 정경과는 달리 하위 게놈 서클, 선형 염색체, 선형 concatamers 및 가지 형 구조 10을 포함하여 organellar 게놈 구조의 더 복잡한 그림에 대한 증거가 증가하고 있습니다. 식물 세포 소기관 게놈의 조립은 상대적으로 큰 크기와 상당한 거꾸로 된 직접 반복에 의해 더욱 복잡해진다. 세포 기관 분리, DNA 정제 및 후속 유전체에 대한 기존의 프로토콜<html> 전자 시퀀싱은 종종 성가 시며 많은 양의 조직을 필요로하며 수백 g의 어린 잎 조직을 출발점 11 , 12 , 13 , 14 , 15 , 16 , 17로 필요로 합니다. 이렇게하면 조직이 제한되어있을 때 세포 내 게놈 시퀀싱을 사용할 수 없게됩니다. 어떤 상황에서는, 세대 간 (through generational basis) 또는 교차점을 통해 유지되어야하는 수컷 멸균 계통에서 필요할 때와 같이, 종자의 양은 제한적이다. 이러한 상황에서 세포 내 소기관 DNA를 정제 한 다음 전체 게놈 증폭을 수행 할 수 있습니다. 그러나 전체 게놈 증폭은 구조적 변이, 하위 게놈 구조 및 이종성 수준을 평가할 때 특히 중요한 문제인 시퀀싱 바이어스를 도입 할 수 있습니다&gt; 18. 짧은 읽기 시퀀싱 기술에 대한 라이브러리 준비의 최근 발전은 전체 게놈 증폭을 피하기 위해 낮은 입력 장벽을 극복했습니다. 예를 들어, Illumina Nextera XT 라이브러리 준비 키트는 1 ng의 적은 DNA를 입력으로 사용할 수 있습니다 19 . 그러나 PacBio 또는 Oxford Nanopore 시퀀싱 기술과 같은 장시간 시퀀싱 응용 프로그램을위한 표준 라이브러리 준비에는 여전히 많은 양의 입력 DNA가 필요하며 이는 세포 내 게놈 시퀀싱에 대한 도전이 될 수 있습니다. 최근에, 입력량을 줄이고 마이크로 그램 - 양의 DNA를 얻기가 어려운 시료에서 게놈 시퀀싱을 용이하게하기 위해 사용자가 작성한 긴 판독 시퀀싱 프로토콜이 개발되었습니다 20 , 21 . 그러나, 고 분자량의 순수한 세포 내 소 분획을 이들 라이브러리 제제에 공급하는 것은 여전히 ​​어려운 과제이다. 우리는o 전체 게놈 증폭없이 NGS에 적합한 세포 내 DNA 농축 및 분리 방법을 비교 및 ​​최적화합니다. 구체적으로, 우리의 목표는 잎의 하위 샘플과 같이 제한된 출발 물질로부터 고 분자량 세포 기관 DNA를 풍부하게하는 우수 사례를 결정하는 것이 었습니다. 이 연구는 organellar DNA를 풍부하게하는 방법의 비교 분석을 제시한다 : (1) 상업적으로 이용 가능한 DNA CpG- 메틸 결합 도메인 단백질 풀다운 접근의 사용에 기초한 DNA 분획 프로토콜 (2) 수정 된 전통적인 분별 원심 분리 프로토콜 22 식물 조직에 적용 23 . 우리는 다른 식물 및 조직 유형으로 쉽게 확장 될 수있는 밀엽 조직으로부터 세포 기관 DNA를 분리하는 최상의 방법을 권장합니다.

<table><tbody><tr><td>2-mercaptoethanol (beta-mercaptoethanol; BME)</td><td>Sigma Aldrich</td><td>M3148-100ml</td><td></td></tr><tr><td>2-propanol (Isopropyl alcohol/isopropanol), bioreagent</td><td>Sigma Aldrich</td><td>I9516</td><td></td></tr><tr><td>agarose, Bio-Rad Cetified Megabase agarose</td><td>Bio-Rad</td><td>1613108</td><td></td></tr><tr><td>analytical balance</td><td>Mettler Toledo</td><td>AB54-S</td><td></td></tr><tr><td>balance</td><td>Mettler Toledo</td><td>PB1502-S</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>B4287-25G</td><td></td></tr><tr><td>Ceramic grinding cylinders, 3/8in x 7/8in</td><td>SPEX SamplePrep</td><td>2183</td><td></td></tr><tr><td>Cryogenic Blocks compatible with tissue homogenizer for holding 50 mL tubes</td><td>SPEX SamplePrep</td><td>2664</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>ethanol, absolute</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA), 0.5 M Solution, pH 8.0</td><td>Fisher</td><td>BP2482-500</td><td></td></tr><tr><td>gel imaging system</td><td></td><td></td><td></td></tr><tr><td>gel stain</td><td></td><td></td><td>Such as GelRed or Ethidium Bromide</td></tr><tr><td>grinding pestle, wide tip for 2 mL conical tubes</td><td></td><td></td><td></td></tr><tr><td>Guanidine-HCl, 8 M solution</td><td>ThermoFisher</td><td>24115</td><td></td></tr><tr><td>LightCycler 480 SYBR Green I Master</td><td>Roche</td><td>4707516001</td><td></td></tr><tr><td>liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Lysing enzymes from Trichoderma harzianum</td><td>Sigma</td><td>L1412</td><td></td></tr><tr><td>Magnesium Chloride</td><td>G Bioscience</td><td>24115</td><td></td></tr><tr><td>magnetic rack</td><td>ThermoFisher</td><td>A13346</td><td></td></tr><tr><td>microcentrifuge tubes, LoBind 1.5 mL&amp;nbsp;</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>microcentrifuge tubes, standard nuclease-free 1.5 mL</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>microcentrifuge, refrigerated</td><td>Sorvall&amp;nbsp;</td><td>Legend X1R</td><td>Or equivalent product, must be capable of reaching at least 18,000 x g with rotors for 50 mL tubes, Oak Ridge tubes, and 1.5 mL tubes</td></tr><tr><td>microcentrifuge, room temperature</td><td>Eppendorf</td><td>5424</td><td>Or equivalent product, must be capable of reaching at least 18,000 x g with rotor for 1.5 mL and 2 mL microcentrifuge tubes</td></tr><tr><td>Microcon DNA Fast Flow Centrifugal Filter Units</td><td>EMD Millipore</td><td>MRCFOR100</td><td></td></tr><tr><td>Miracloth, 1 square per sample cut to fit funnel</td><td>EMD Millipore</td><td>475855</td><td></td></tr><tr><td>NEBNext Microbiome DNA Enrichment Kit</td><td>New England Biolabs</td><td>E2612L</td><td></td></tr><tr><td>parafilm</td><td>Parafilm M</td><td>PM992</td><td></td></tr><tr><td>plastic pots and trays</td><td></td><td></td><td></td></tr><tr><td>polyvinylpyrrolidone (PVP)</td><td>Fisher</td><td>BP431-100</td><td></td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>19131</td><td></td></tr><tr><td>Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III)</td><td>Bio-Rad</td><td>1703697</td><td></td></tr><tr><td>purification beads, Agencourt AMpureXP beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>QIAamp DNA Mini Kit</td><td>Qiagen</td><td>51304</td><td></td></tr><tr><td>Qiagen 20/g Genomic Tip DNA Extraction Kit</td><td>Qiagen</td><td>10223</td><td></td></tr><tr><td>Qiagen Buffer EB (elution buffer)</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>Qiagen DNA Extraction Buffer Set</td><td>Qiagen</td><td>19060</td><td></td></tr><tr><td>QiaRack</td><td>Qiagen</td><td>19015</td><td></td></tr><tr><td>qPCR machine (e.g. Roche Light Cycler 480)</td><td>Roche</td><td></td><td></td></tr><tr><td>qPCR plate sealing film</td><td>Roche</td><td>4729757001</td><td></td></tr><tr><td>qPCR plate, 96 well plate</td><td>Roche</td><td>4729692001</td><td></td></tr><tr><td>Qubit&amp;nbsp;assay tubes</td><td>Life Technologies</td><td>Q32856</td><td></td></tr><tr><td>Qubit Broad Spectrum assay kit</td><td>Life Technologies</td><td>Q32850</td><td></td></tr><tr><td>Qubit High Sensitivity assay kit</td><td>Life Technologies</td><td>Q32851</td><td></td></tr><tr><td>RNaseA</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Serological pipettes (20 mL) and pipet-aid</td><td>Fisher</td><td>13-678-11</td><td></td></tr><tr><td>Small funnels, 1 per sample</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Ambion</td><td>AM9759</td><td></td></tr><tr><td>Soft paintbrush, 2 per sample</td><td></td><td></td><td></td></tr><tr><td>SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer</td><td>SPEX SamplePrep</td><td></td><td>Or another comparable tissue homogenizer. If you do not have access to a tissue homogenizer, then grinding in a pre-chilled mortar and pestle will suffice (see protocol for details). However, a homogenizer will give more consistent results and total homogenization time is reduced.</td></tr><tr><td>Sucrose</td><td>Omnipure</td><td>8550</td><td></td></tr><tr><td>TBE</td><td></td><td></td><td></td></tr><tr><td>thermomixer</td><td></td><td></td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td>T2819-100ml</td><td></td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td></td></tr><tr><td>tube rotater</td><td></td><td></td><td></td></tr><tr><td>tubes, 50 mL conical polypropylene</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>tubes, 50 mL high-speed polypropylene&amp;nbsp;</td><td>ThermoScientific/Nalgene</td><td>3119-0050</td><td>e.g. Nalgene Oakridge tubes or equivalent</td></tr><tr><td>vermiculite</td><td></td><td></td><td></td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr><tr><td>water, sterile and certified Nuclease-free&amp;nbsp;</td><td>Fisher</td><td>1481</td><td></td></tr><tr><td>water, sterile milliQ</td><td></td><td></td><td></td></tr></tbody></table>

optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing

식물 세포 소기관 게놈은 복잡한 구조 및 / 또는 서브 게놈 조각을 형성하기 위해 페어링 또는 재조합을 거칠 수있는 크고 반복적 인 요소를 포함합니다. Organellar 게놈은 또한 주어진 세포 또는 조직 유형 (heteroplasmy) 내의 혼합물에 존재하며, 아형의 다량은 발육 기간 동안 또는 스트레스하에있을 때 (부분 화학량 론적 이동) 변화 할 수있다. 세포 기관 게놈 구조와 기능에 대한 더 깊은 이해를 위해서는 차세대 시퀀싱 (NGS) 기술이 필요합니다. 전통적인 sequencing 연구는 organellar DNA를 얻기 위해 몇 가지 방법을 사용합니다 : (1) 대량의 시작 조직이 사용되면 균질화되고 차등 원심 분리 및 / 또는 기울기 정화가 이루어집니다. (종자, 재료 또는 공간이 한정되어있는 경우, 즉) (2) 조직의 작은 양이 사용된다면, 동일한 프로세스는 (1) 충분한 DNA를 얻기 위해 전체 게놈 증폭 다음과 같이 수행된다. (3) 생물 정보학 분석은 seq전체 게놈 DNA를 분석하고 세포 기관 판독을 분석합니다. 이러한 모든 방법에는 고유 한 문제점과 단점이 있습니다. (1)에서 이와 같이 많은 양의 시작 조직을 얻는 것이 어려울 수 있습니다. (2)에서, 전체 게놈 증폭은 시퀀싱 바이어스를 도입 할 수있다; (3)에서 핵과 세포 내 게놈 간의 상동 성은 집합과 분석을 방해 할 수있다. 거대한 핵 게놈을 가진 식물에서, organellar DNA를 풍부하게하여 생물 정보학 분석을위한 서열화 비용 및 서열 복잡성을 줄이는 것이 유리하다. 여기에서 전통적인 차동 원심 분리 방법과 네 번째 방법 인 CpG- 메틸 풀다운 방식을 비교하여 총 게놈 DNA를 핵 및 세포 내 분획으로 분리합니다. 두 가지 방법 모두 미토콘드리아와 엽록체의 비율이 다르긴하지만 세포 내 소기관 서열에 대해 고도로 농축 된 DNA 인 NGS에 충분한 양의 DNA를 생산합니다. 우리는 밀 잎 조직을위한 이러한 방법의 최적화를 제시하고 주요 이점과 d샘플 입력, 프로토콜 용이성 및 다운 스트림 응용 프로그램의 맥락에서 각 접근법의 단점.

이 원고에 제시된 프로토콜은 식물 조직에서 세포 기관 DNA를 풍부하게하는 두 가지 방법을 설명합니다. 여기에 제시된 조건은 밀의 조직 최적화를 반영합니다. 프로토콜의 주요 단계, 필요한 조직 입력 및 DNA 출력의 비교는 그림 1에 설명되어 있습니다. 우리가 테스트 한 DC 프로토콜의 단계는 이전에 설명한 것과 비슷한 조건을 따른다 ( <strong class="x...

Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

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

2 가지 plant organellar DNA 농축 방법의 비교 및 ​​최적화가 제시됩니다 : 전통적인 차동 원심 분리 및 메틸화 상태에 기초한 총 gDNA 분획. 우리는 결과로 나오는 DNA 양과 품질을 평가하고, 짧은 읽기 차세대 시퀀싱에서의 성능을 시연하며, 장시간 읽기 단일 분자 시퀀싱에서의 사용 가능성에 대해 논의합니다.

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 ...

optimization-comparative-analysis-plant-organellar-dna-enrichment

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

12.8K 조회수.  United States Department of Agriculture-Agricultural Research Service.  2 가지 plant organellar DNA 농축 방법의 비교 및 ​​최적화가 제시됩니다 : 전통적인 차동 원심 분리 및 메틸화 상태에 기초한 총 gDNA 분획. 우리는 결과로 나오는 DNA 양과 품질을 평가하고, 짧은 읽기 차세대 시퀀싱에서의 성능을 시연하며, 장시간 읽기 단일 분자 시퀀싱에서의 사용 가능성에 대해 논의합니다. 

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

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 ...

연구

유전학

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

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 a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

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

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 ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

다음-세대 시퀀싱과 생물 정보학 파이프라인 헌법 질병의 유전 결정 요인 평가 대상

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

루트 미생물 탐색: 토양, Rhizosphere, 및 루트 Endosphere에서 세균성 지역 사회 데이터 추출

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

저하된 FFPE-RNA 시료의 시퀀싱 및 분석을 위한 최적화

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

<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 ...

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.

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 ...

의학

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.

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱

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

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

긴 읽기 시퀀싱에 대한 흰가루병에서 높은 분자량의 게놈의 DNA의 정제

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

다음-세대 시퀀싱과 생물 정보학 파이프라인 헌법 질병의 유전 결정 요인 평가 대상

루트 미생물 탐색: 토양, Rhizosphere, 및 루트 Endosphere에서 세균성 지역 사회 데이터 추출

저하된 FFPE-RNA 시료의 시퀀싱 및 분석을 위한 최적화

약용 식물을 식별하기 위한 DNA 바코드 적용

약용 식물을 식별하기 위한 DNA 바코드 적용

약용 식물을 식별하기 위한 DNA 바코드 적용

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱