Name: robust dna isolation and high-throughput sequencing library construction for herbarium specimens
Uploaded: 2018-03-08T14:30:00
Description: Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

Taylor AuBuchon-yaşlı, Jordan Teisher ve Kristina Zudock için örnekleme sulak numuneler yardım ve Missouri Botanical Garden erişim sulak numuneler için yıkıcı örnekleme için teşekkür ederiz. Bu eser Ulusal Bilim Vakfı (DEB-1457748) bir hibe destek oldu.

1. başlangıç önce
<ol><li>CTAB, polyvinylpyrrolidone (PVP) 40, 100,0 mL 1 M Tris pH 8.0, 0, 5 M ethylenediaminetetraacetic asit (EDTA) pH 8.0, 280,0 mL 40 mL 5 10 g 20 g ekleyerek taze setil trimethylammonium bromür (CTAB) arabellek17 olun M NaCl ve 400.0 mL reaktif su birlikte, Toplam hacim 1 L reaktif sınıf kullanarak su getir. 8,0 pH ayarlayın. Not: Ek reaktifler CTAB için bireysel takson olarak ikincil bileşikler bağlı olarak ilave edilebilir. Allen ve ark. görmek <s...

<ol>
	<li>Savolainen, V., Cu&#233;noud, P., Spichiger, R., Martinez, M. D. P., Cr&#232;vecoeur, M., Manen, J. 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+use+of+herbarium+specimens+in+DNA+phylogenetics:+Evaluation+and+improvement.">The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement.</a> Plant Syst Evo. 197 (1-4), 87-98 (1995).</li><li>Zedane, L., Hong-Wa, C., Murienne, J., Jeziorski, C., Baldwin, B. G., Besnard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Museomics+illuminate+the+history+of+an+extinct,+paleoendemic+plant+lineage+(Hesperelaea,+Oleaceae)+known+from+an+1875+collection+from+Guadalupe+Island,+Mexico.">Museomics illuminate the history of an extinct, paleoendemic plant lineage (Hesperelaea, Oleaceae) known from an 1875 collection from Guadalupe Island, Mexico.</a> Bio J Linn Soc. 117 (1), 44-57 (2016).</li><li>Teisher, J. K., McKain, M. R., Schaal, B. A., Kellogg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphyly+of+Arundinoideae+(Poaceae)+and+Evolution+of+the+Twisted+Geniculate+Lemma+Awn.">Polyphyly of Arundinoideae (Poaceae) and Evolution of the Twisted Geniculate Lemma Awn.</a> Ann Bot. , (2017).</li><li>Cozzolino, S., Cafasso, D., Pellegrino, G., Musacchio, A., Widmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+time+and+space:+the+use+of+herbarium+specimens+to+reconstruct+patterns+of+genetic+variation+in+the+endangered+orchid+Anacamptis+palustris.">Genetic variation in time and space: the use of herbarium specimens to reconstruct patterns of genetic variation in the endangered orchid Anacamptis palustris.</a> Conserv Gen. 8 (3), 629-639 (2007).</li><li>Wandeler, P., Hoeck, P. E. A., Keller, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+museum+specimens+in+population+genetics.">Back to the future: museum specimens in population genetics.</a> Tre Eco &amp; Evo. 22 (12), 634-642 (2007).</li><li>Rivers, M. C., Taylor, L., Brummitt, N. A., Meagher, T. R., Roberts, D. L., Lughadha, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+herbarium+specimens+are+needed+to+detect+threatened+species?.">How many herbarium specimens are needed to detect threatened species?.</a> Bio Conserv. 144 (10), 2541-2547 (2011).</li><li>Saltonstall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptic+invasion+by+a+non-native+genotype+of+the+common+reed,+Phragmites+australis,+into+North+America.">Cryptic invasion by a non-native genotype of the common reed, Phragmites australis, into North America.</a> PNAS USA. 99 (4), 2445-2449 (2002).</li><li>Besnard, 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=From+museums+to+genomics:+old+herbarium+specimens+shed+light+on+a+C3+to+C4+transition.">From museums to genomics: old herbarium specimens shed light on a C3 to C4 transition.</a> J Exp Bot. 65 (22), 6711-6721 (2014).</li><li>S&#228;rkinen, T., Staats, M., Richardson, J. E., Cowan, R. S., Bakker, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+open+the+treasure+chest?+Optimising+DNA+extraction+from+herbarium+specimens.">How to open the treasure chest? Optimising DNA extraction from herbarium specimens.</a> PLoS ONE. 7 (8), e43808 (2012).</li><li>Harris, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+analysis+of+tropical+plant+species:+An+assessment+of+different+drying+methods.">DNA analysis of tropical plant species: An assessment of different drying methods.</a> Plant Syst Evo. 188 (1-2), 57-64 (1994).</li><li>Staats, 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=DNA+damage+in+plant+herbarium+tissue.">DNA damage in plant herbarium tissue.</a> PLoS ONE. 6 (12), e28448 (2011).</li><li>Bakker, F. T., 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=Herbarium+genomics:+plastome+sequence+assembly+from+a+range+of+herbarium+specimens+using+an+Iterative+Organelle+Genome+Assembly+pipeline.">Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly pipeline.</a> Bio J of the Linn Soc. 117 (1), 33-43 (2016).</li><li>Camacho, F. J., Gernandt, D. S., Liston, A., Stone, J. K., Klein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endophytic+fungal+DNA,+the+source+of+contamination+in+spruce+needle+DNA.">Endophytic fungal DNA, the source of contamination in spruce needle DNA.</a> Mol Eco. 6 (10), 983-987 (1997).</li><li>Hofreiter, M., Jaenicke, V., Serre, D., Von Haeseler, A., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequences+from+multiple+amplifications+reveal+artifacts+induced+by+cytosine+deamination+in+ancient+DNA.">DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.</a> Nucl Acids Res. 29 (23), 4793-4799 (2001).</li><li>Staats, 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=Genomic+treasure+troves:+Complete+genome+sequencing+of+herbarium+and+insect+museum+specimens.">Genomic treasure troves: Complete genome sequencing of herbarium and insect museum specimens.</a> PLoS ONE. 8 (7), e69189 (2013).</li><li>Bakker, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbarium+genomics:+skimming+and+plastomics+from+archival+specimens.">Herbarium genomics: skimming and plastomics from archival specimens.</a> Webbia. 72 (1), 35-45 (2017).</li><li>Doyle, J. J., Doyle, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+DNA+isolation+procedure+for+small+quantities+of+fresh+leaf+tissue.">A rapid DNA isolation procedure for small quantities of fresh leaf tissue.</a> Phytochem Bul. 19, 11-15 (1987).</li><li>Allen, G. C., Flores-Vergara, M. A., Krasynanski, S., Kumar, S., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+protocol+for+rapid+DNA+isolation+from+plant+tissue+using+cetryltrimethylammonium+bromide.">A modified protocol for rapid DNA isolation from plant tissue using cetryltrimethylammonium bromide.</a> Nat Prot. 1, 2320-2325 (2006).</li><li>Twyford, A. D., Ness, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+complete+plastid+genome+seqeuncing.">Strategies for complete plastid genome seqeuncing.</a> Mol Eco Resour. , (2016).</li><li>Aird, D., 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=Analyzing+and+minimizing+PCR+amplification+bias+in+Illumina+sequencing+libraries.">Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.</a> Genome Bio. 12 (2), R18 (2011).</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> Bioinf. 30, 2114-2120 (2014).</li><li>Grigoriev, I. V., 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=MycoCosm+portal:+gearing+up+for+1000+fungal+genomes.">MycoCosm portal: gearing up for 1000 fungal genomes.</a> Nucl Acids Res. 42 (1), D699-D704 (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 Meth. 9 (4), 357-359 (2012).</li><li>Herbarium Genomics. Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>. Fast-Plast: Rapid de novo assembly and finishing for whole chloroplast genomes Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>McKain, M. R., McNeal, J. R., Kellar, P. R., Eguiarte, L. E., Pires, J. C., Leebens-Mack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+rapid+diversification+and+convergent+origins+of+active+pollination+within+Agavoideae+(Asparagaceae).">Timing of rapid diversification and convergent origins of active pollination within Agavoideae (Asparagaceae).</a> Am J Bot. 103 (10), 1717-1729 (2016).</li><li>McKain, M. R., Hartsock, R. H., Wohl, M. M., Kellogg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verdant:+automated+annotation,+alignment,+and+phylogenetic+analysis+of+whole+chloroplast+genomes.">Verdant: automated annotation, alignment, and phylogenetic analysis of whole chloroplast genomes.</a> Bioinf. , (2016).</li><li>Staton, S. E., Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposome:+A+toolkit+for+annotation+of+transposable+element+families+from+unassembled+sequence+reads.">Transposome: A toolkit for annotation of transposable element families from unassembled sequence reads.</a> Bioinf. 31 (11), 1827-1829 (2015).</li><li>Bao, W., Kojima, K. K., Kohany, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repbase+Update,+a+database+of+repetitive+elements+in+eukaryotic+genomes.">Repbase Update, a database of repetitive elements in eukaryotic genomes.</a> Mobile DNA. 6 (1), 11 (2015).</li><li>. Transposons Available from: <a target="_blank" href="https://github.com/mrmckain/">https://github.com/mrmckain/</a> (2017)</li><li>Wei&#223;, C. L., 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=Temporal+patterns+of+damage+and+decay+kinetics+of+DNA+retrieved+from+plant+herbarium+specimens.">Temporal patterns of damage and decay kinetics of DNA retrieved from plant herbarium specimens.</a> Royal Soc Open Sci. 3 (6), 160239 (2016).</li><li>Sawyer, S., Krause, J., Guschanski, K., Savolainen, V., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+patterns+of+nucleotide+misincorporations+and+DNA+fragmentation+in+ancient+DNA.">Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.</a> PLoS ONE. 7 (3), e34131 (2012).</li><li>Head, S. 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=Library+construction+for+next-generation+sequencing:+overviews+and+challenges.">Library construction for next-generation sequencing: overviews and challenges.</a> BioTechniques. 56 (2), 61-64 (2014).</li><li>Grover, C. E., Salmon, A., Wendel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sequence+capture+as+a+powerful+tool+for+evolutionary+analysis.">Targeted sequence capture as a powerful tool for evolutionary analysis.</a> Am J Bot. 99, 312-319 (2012).</li></ol>

Burada sunulan Protokolü DNA izolasyon ve Kütüphane hazırlık kurutulmuş bitki örnekleri üzerinden sıralama için kapsamlı ve sağlam bir yöntemdir. Yöntemi ve bunu değiştirmek için çok az ihtiyaç örnek kalite make büyük sulak alan sıralama projeler için ölçeklenebilir temel. Düşük verim kitaplıkları için bir isteğe bağlı reamplification adım dahil düşük kaliteli, düşük miktar, nadir, ya da başka türlü sıralama için uygun olmaz tarihsel açıdan önemli örnekleri dahil sağlar...

Sulak koleksiyonları tür ve genomik çeşitlilik filogenetik1,2,3, Populasyon genetiği4,5, koruma çalışmaları için potansiyel olarak değerli bir kaynaktır Biyoloji6, istilacı türlerin Biyoloji7ve özellik evrim8. Zengin bir çeşitlilik türlerin, nüfus, coğrafi yer ve zaman puan elde etmek için yeteneğini sulak olduğunu "hazine sandığı"9 vurgulamaktadır. Tarihsel olarak, bozulmuş doğa sulak elde edilen DNA PCR tabanlı projeler genellikle yalnızca bölgeleri kloroplast genom veya ribozomal, iç kopya etmek Rondela (ITS) gibi yüksek bir kopyasını buldum işaretçileri kullanarak araştırmacılar relegating engel RNA. Örnekler ve DNA kalitesini kapsamlı koruma9,10, çift iplikçikli sonları ve parçalanma hasar en yaygın formları olmak, oluşturma kurutma işleminde kullanılan ısı ile yöntemleri göre değişir sözde % 90'ı DNA kilit-up bu çalışmalar PCR tabanlı11ipotekli. Parçalanma bir yana, ikinci en yaygın sulak genomik kirlenme, bu endophytic mantarlar13 türetilmiş veya mantar toplama sonra ama yine de içinde sulak12, montaj önce öldükten sonra edinilen gibi sorundur Bu sorun çözüldü bioinformatically şu mantar veritabanı (aşağıya bakın) verilen olabilir. Üçüncü ve daha az yaygın bir sorun ile sitozin Deaminasyon (C/G→T/A)14, sıra değişiklik olsa da sulak numuneler11' (~ %0,03) düşük olduğu tahmin edilmektedir. Yüksek işlem hacmi sıralama (HTS) gelişiyle, parçalanma sayısında kısa okuma ve düşük kalitesi ile çok sayıda numune üzerinden genomik düzeyinde veri toplama izin sıralama derinliği12,15, üstesinden gelebilir DNA ve hatta bazen tüm genom sıralama15erişimine izin verme.
Sulak örnekleri daha sık kullanılan olma ve filogenetik projeleri16büyük bir bileşenidir. HTS için sulak örnekler kullanarak geçerli bir meydan okuma tutarlı bir şekilde yeterli çift telli DNA, sıralama iletişim kuralları için gerekli bir ön koşul zamanında, çok sayıda tür yöntemleri birey için optimize etmek gerek kalmadan almaktır numuneler. Bu yazıda, bu mevcut yöntemlerden yararlanır ve onlara hızlı ve yinelenebilir sonuçları için izin vermek için değiştirir DNA ekstraksiyon ve Kütüphane sulak numunelerin hazırlanması için bir iletişim kuralı gösterilmiştir. Bu yöntem için tam 13 h, 8 h uygulamalı zamanla veya isteğe bağlı reamplification adım gerekli olduğunda 9 h uygulamalı zamanla 16 h 24 örnekleri kitaplığına örnek işleme sağlar. Daha fazla örnek aynı anda işleme ulaşılabilir, olsa da santrifüj kapasitesi ve teknik beceri sınırlayıcı faktördür. Protokol yalnızca tipik labaratuar donanımları (thermocycler, santrifüj ve manyetik duruyor) Nebulizatör veya sonicator, gibi özel ekipman yerine DNA kesme için gerektirecek şekilde tasarlanmıştır.
DNA kalite, parça boyutu ve miktar faktörler sulak örneklerin yüksek üretilen iş sıralama deneylerde kullanmak için sınırlı hale gelir. Sulak DNA izole ve yüksek işlem hacmi sıralama kitaplıkları oluşturmak için diğer yöntemleri 10 kadar az kullanma belgili tanımlık yarar göstermiştir ng DNA16; Ancak onlar deneysel olarak PCR optimum sayısını belirleme gerektiren bir kütüphane hazırlık için gerekli geçer. Son derece küçük miktarlarda uygun çift ile ilgili DNA (dsDNA), telli birkaç sulak numune yalnızca bir tek kitaplık hazırlık için yeterli DNA üretmek gibi bu pratik olur. DNA yok Kütüphane optimizasyonu adımda kayıp burada sunulan yöntem döngüleri örnek kalite, ne olursa olsun tek bir sayı kullanır. Bunun yerine, kitaplıkları sıralama için gerekli minimum tutarları uygun olmayan bir reamplification adım çağrılır. Birçok sulak örnekleri nadirdir ve sahip küçük malzeme birçok durumda yıkıcı örnekleme haklı zorlaştırır. Bu karşı sunulan Protokolü sağlar dsDNA giriş boyutları daha az 1,25 ng Kütüphane hazırlık sürecine yüksek üretilen iş sıralama ve örneklerin yıkıcı örnekleme gereksinimini en aza indirilmesi için uygun örnekleri kapsamını genişletiyor.
Aşağıdaki iletişim kuralı otlar için en iyi duruma getirilmiş ve protokol başka birçok bitki gruplarına uygulanabilir beklemek rağmen sulak örnekleri, farklı türlerin yüzlerce test. Düşük kaliteli ve/veya nadir örnekleri kaydetmek için kullanılan bir isteğe bağlı kurtarma adım içerir. Test iki yüz sulak numuneler üzerinde bağlı olarak, bu protokol bırakmak nadir örnekleri minimal yıkıcı örnekleme yoluyla korunması için numuneler ile düşük doku girdi ve kalite, çalışır. Burada bu protokolü phylogenomics tabanlı projeler için sıralı kaliteli kitaplıkları sağlayabilir gösterilir.

<table border="0" cellpadding="0" cellspacing="0" width="733">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Veriti Thermal Cycler</td>
			<td style="width:180px;">Applied Biosystems</td>
			<td style="width:104px;">4452300</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Imaging System</td>
			<td style="width:180px;">Azure Biosystems</td>
			<td style="width:104px;">c300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microfuge 20 Series</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">B30137</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Digital Dry Bath</td>
			<td style="width:180px;">Benchmark Scientific</td>
			<td style="width:104px;">BSH1001</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Electrophoresis System</td>
			<td style="width:180px;">EasyCast</td>
			<td style="width:104px;">B2</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PURELAB flex 2 (Ultra pure water)</td>
			<td style="width:180px;">ELGA&#160;</td>
			<td style="width:104px;">89204-092</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DNA LoBind Tube&#160;</td>
			<td style="width:180px;">Eppendorf</td>
			<td>30108078</td>
			<td style="width:147px;">2 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mini centrifuge</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-006-901</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vortex-Genie 2</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">12-812</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mortar</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">S02591</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pestle</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">S02595</td>
			<td style="width:147px;">porcelain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge tubes</td>
			<td style="width:180px;">fisher Scientific</td>
			<td style="width:104px;">21-403-161</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microwave</td>
			<td style="width:180px;">Kenmore</td>
			<td style="width:104px;">405.7309231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit Assay Tubes</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">0.2 ml Strip tube and Cap for PCR</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">20170-004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit 2.0 Fluorometer</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:104px;">Q32866</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Balance</td>
			<td style="width:180px;">Mettler Toledo</td>
			<td style="width:104px;">PM2000</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen Short-term Storage</td>
			<td style="width:180px;">Nalgene</td>
			<td style="width:104px;">F9401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Magnetic-Ring Stand&#160;</td>
			<td style="width:180px;">ThermoFisher Scientific&#160;</td>
			<td style="width:104px;">AM10050</td>
			<td style="width:147px;">96 well&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Water Bath</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">89032-210</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hot Plate Stirrers</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">97042-754</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Liquid Nitrogen</td>
			<td style="width:180px;">Airgas</td>
			<td style="width:104px;">UN1977</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1 X TE Buffer</td>
			<td style="width:180px;">Ambion</td>
			<td style="width:104px;">AM9849</td>
			<td style="width:147px;">pH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CTAB</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0833-500G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2-MERCAPTOETHANOL</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">0482-200ML</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ribonuclease A</td>
			<td style="width:180px;">AMRESCO</td>
			<td style="width:104px;">E866-5ML</td>
			<td style="width:147px;">10 mg/ml solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Agencourt AMPure XP</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:104px;">A63882</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Chloride</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">705744</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isopropyl Alcohol</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">40970004-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nuclease Free water</td>
			<td style="width:180px;">bio WORLD</td>
			<td style="width:104px;">42300012-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoamyl Alcohol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">A393-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium Acetate Trihydrate</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:104px;">s608-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">LE Agarose</td>
			<td style="width:180px;">GeneMate</td>
			<td style="width:104px;">E-3120-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">100bp PLUS DNA Ladder</td>
			<td style="width:180px;">Gold Biotechnology</td>
			<td style="width:104px;">D003-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA, Disodium Salt</td>
			<td style="width:180px;">IBI Scientific</td>
			<td style="width:104px;">IB70182</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:104px;">Q32854</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TRIS</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td style="width:104px;">103133</td>
			<td style="width:147px;">ultra pure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gel Loading Dye Purple (6 X)</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">B7024S</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext dsDNA Fragmentase</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0348L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Ultra II DNA Library Prep Kit for Illumina&#160;</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7645L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">NEBNext Multiplex Oligos for Illumina</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">E7600S</td>
			<td style="width:147px;">Dual Index Primers Set 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NEBNext Q5 Hot Start HiFi PCR Master Mix</td>
			<td style="width:180px;">New England BioLabs</td>
			<td style="width:104px;">M0543L</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mag-Bind RXNPure Plus</td>
			<td style="width:180px;">Omega bio-tek</td>
			<td style="width:104px;">M1386-02</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">GelRed 10000 X</td>
			<td style="width:180px;">Pheonix Research</td>
			<td style="width:104px;">41003-1</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Phenol solution</td>
			<td style="width:180px;">SIGMA Life Science</td>
			<td style="width:104px;">P4557-400ml</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PVP40</td>
			<td style="width:180px;">SIGMA-Aldrich</td>
			<td style="width:104px;">PVP40-50G</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Chloroform</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">EM8.22265.2500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ethanol</td>
			<td style="width:180px;">Koptec</td>
			<td style="width:104px;">V1016</td>
			<td style="width:147px;">200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Silica sand</td>
			<td style="width:180px;">VWR</td>
			<td style="width:104px;">14808-60-7</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reamplification primers</td>
			<td colspan="2">Integrated DNA Technologies</td>
			<td>see text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sequencher v.5.0.1</td>
			<td style="width:180px;">GeneCodes</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Herbaria biyolojik çalışmalar çeşitli kullanılan bitki materyali paha biçilmez bir kaynaktır. Sulak örnekler kullanımı zorlukları örnek koruma kalitesi, bozulmuş DNA ve nadir örneklerin yıkıcı örnekleme dahil olmak üzere, bir dizi ile ilişkilidir. Daha etkili bir şekilde sulak malzeme büyük sıralama projelerde kullanmak için güvenilir ve ölçeklenebilir yöntemi DNA izolasyon ve Kütüphane hazırlanması gereklidir. Bu kağıt bir sağlam, başına sonuna protokol değişikliği için bireysel örnekleri gerektirmeyen DNA izolasyon ve yüksek işlem hacmi Kütüphane yapımı için sulak örnekler üzerinden gösteriyor. Bu iletişim kuralı, Kütüphane boyutu seçimi değiştirme ve düşük verim kitaplıkları için bir isteğe bağlı reamplification adım tanıtımı doku kabuğu optimize ederek düşük kaliteli kurutulmuş bitki malzeme ve alır avantajı mevcut yöntemler için hazırlanmıştır. Reamplification düşük verim DNA kütüphanelerinin yeri doldurulamaz ve potansiyel olarak değerli sulak numuneler, ihtiyaç için ek yıkıcı örnekleme ve fark edilebilir sıralama önyargı için ortak tanıtımı olmadan inkâr türetilen örnekleri kurtarabilirsiniz Filogenetik uygulamaları. Protokol çim türleri yüzlerce üzerinde test edilmiştir, ancak doğrulama sonra diğer bitki soy kullanmak için adapte olması bekleniyor. Bu iletişim kuralı nerede parçaları istediğiniz boyuta aralığında var olmayan, son derece bozulmuş DNA ve temiz DNA izolasyon inhibe Sekonder metabolitler bazı bitki malzeme mevcut sınırlı olabilir. Genel olarak, bu protokol bırakmak için DNA izolasyon ve az 13 h, 24 örnekleri kitaplığı hazırlanması sadece 8 h minimal değişiklikler ile aktif uygulamalı zaman ile hızlı ve kapsamlı bir yöntem sağlar.

DNA izolasyonu ve son Kütüphane verim Bu çalışmada, sulak DNA izolasyonu ve yüksek kaliteli sıralama kitaplıkları kurtarılması için iletişim kuralı etkinliğinin 1920 en eski ve en küçük 2012 (Tablo 2) ile 50 farklı örnekleri kullanarak gösterilmiştir. Her örnek için yaklaşık 10 mg yaprak doku DNA izolasyon için kullanıldı. Yeşil yaprak dokusu varsa tercih ve hiçbir doku bariz mantar kirlenme ile seçilmiştir. Verim düş...

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

Sağlam DNA izolasyon ve yüksek işlem hacmi sıralama kitaplığı inşaat sulak numuneler için

Bu makalede, DNA izolasyon ve yüksek işlem hacmi sıralama kitaplığı inşaat sulak malzeme son derece kalitesiz DNA'ın kurtarma dahil olmak üzere ayrıntılı bir protokol gösterilir.

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 ...

The overall goal of this protocol is to increase the quantity of isolated double-stranded DNA and subsequent high-throughput sequencing libraries from herbarium specimens, maximizing their utility in phylogenomic studies. This method unlocks the potential of herbarium specimens that would otherwise not be useful for sequencing projects as genomic sequences because of extensive sample degradation. The main advantage of this technique is that it is robust enough that rare and historically significant herbarium specimens can be used without the need for extensive destructive sampling.This technique extends the depth of taxon sampling in systematic spaced projects. Species acquisition is a limiting step, but with this protocol, historical collections can be easily used into these studies. Add liquid nitrogen and 30 to 50 milligrams of sterilized sand to grind one centimeter square of preselected herbarium tissue to fine powder in pre-chilled mortar and pestle.Adequate grinding is essential for successful DNA isolation. And care should be taken that the sample is completely pulverized. Next, add the frozen powder to two two-milliliter tubes, filling no more than half of the tube volume.Then add 600 microliters of warm CTAB solution to each tube. Thoroughly mix the powder by inverting and vortexing. Then incubate the samples in a water bath maintained at 65 degrees Celsius for one to 1.5 hours.In the meantime, vortex every 15 minutes. Then centrifuge the samples at 10, 000 g for five minutes at room temperature. After centrifugation, transfer approximately 500 microliters of the supernatant to a new, fresh set of labeled tubes.To the supernatant, add four microliters of 10 milligrams per milliliter of RNase A to each tune. Then mix the contents of the tube by inverting or pipetting. Incubate the samples in a heat block or water bath maintained at 37 degrees Celsius for 15 minutes.Once the tubes have obtained room temperature, add approximately 500 microliters of 25:24:1 phenol, chloroform, isoamyl alcohol mixture to the samples. Then mix the contents of the tubes by inverting or repeated pipetting. Then centrifuge the samples at 12, 000 g for 15 minutes at room temperature.Transfer the upper aqueous layer, which is approximately 400 microliters, to a fresh set of labeled tubes. To the aqueous layer in each tube, add approximately 400 microliters of 24:1 chloroform, isoamyl alcohol mixture. Mix the sample thoroughly by repeated pipetting or inversion.Then centrifuge the tubes at 12, 000 g for five minutes and transfer approximately 300 microliters of upper aqueous layer to pre-chilled, labeled tubes. Next, add 300 microliters of pre-chilled isopropanol and 12 microliters of 2.5 molar sodium acetate. Then incubate the tubes at negative 20 degrees Celsius for 30 to 60 minutes.After incubation, remove the samples from the freezer. Next, centrifuge the samples at 12, 000 g for 15 minutes at room temperature. After centrifugation, wash the pellet with approximately 300 to 500 microliters of 70%ethanol.Again, centrifuge the tubes at 12, 000 g for 10 minutes. Then without disturbing the pellet, carefully remove the supernatant. Next, air-dry the pellets and dissolve the DNA in 50 microliters of 1X Tris-EDTA buffer.Add one to 16 microliters of isolated DNA and two microliters of fragmentation reaction buffer in a 0.2-milliliter polymerase chain reaction tube. Adjust the final volume to 18 microliters with nuclease-free water. Next, add two microliters of double-stranded DNA fragmentation enzyme and vortex the mixture briefly for tree seconds.Then incubate the samples at 37 degrees Celsius for 8.5 minutes in the thermocycler. Post incubation, add five microliters of 0.5 molar EDTA to the tubes. Add 25 microliters of nuclease-free water to adjust the total volume of the sheared DNA to 50 microliters.Then add 45 microliters of SPRI beads maintained at room temperature to 50 microliters of sheared DNA and mix the two thoroughly by pipetting it up and down. Leave the sample containing the tubes for five minutes. Then transfer the tubes on a magnetic plate and wait for another five minutes.Carefully expel the supernatant. Next, add 200 microliters of fresh 80%ethanol to the tubes on the magnetic stand and wait for another 30 seconds at room temperature. After 30 seconds, carefully remove the supernatant.Leave the lid of the tube open to air-dry the beads for five minutes on the magnetic stand. Then remove the tubes from the magnet stand and elute the DNA from the beads in 55 microliters of 0.1X Tris-EDTA buffer. Mix the DNA thoroughly by repeated pipetting.Then incubate the sample at room temperature for five minutes. Again, place the tube on the magnetic stand and wait for the solution to clear in approximately two minutes. Remove 52 microliters of the supernatant and then perform DNA quantification analysis as outlined in the text protocol.To 50 microliters of cleaned and sheared DNA, add three microliters of endonuclease, phosphate tailing enzymes, and seven microliters of reaction buffer. Mix the DNA and the other components thoroughly by pipetting up and down. Then spin the tubes to remove the bubbles.After spinning, transfer the samples to a thermocycler and set the program. Dilute the adaptor 25 to 50-fold to achieve a working concentration of 0.6 to 0.3 micromolar. For high-throughput short-read sequencing, add 30 microliters of ligation master mix, one microliter of ligation enhancer, and 2.5 microliters of adaptor to the tubes.Mix the contents of the tubes thoroughly by pipetting up and down. Again, spin the tubes to remove any bubbles. Then incubate the tube at 20 degrees Celsius for 15 minutes.Add three microliters of commercial mixture of uracil-DNA glycosylase and DNA glycosylase lyase endonuclease VIII to the tubes. Adjust the total volume to 96.5 microliters. Mix the components thoroughly and incubate at 37 degrees Celsius for 15 minutes in the thermocycler.In the tube, prepare the polymerase chain reaction mix by adding all the reagents to cleaned, adaptor-ligated DNA. Then mix the reaction well by vortexing. Once the vortex is over, put the samples in the thermocycler and start the thermocycler amplification setting.Add about 25 microliters of magnetic beads maintained at room temperature to the sample and mix by repeated pipetting. Incubate the sample for five minutes. Then transfer the tubes on a magnetic stand and wait for another five minutes.After five minutes, remove the supernatant to a new set of labeled tubes. Next, to the supernatant, add six microliters of SPRI beads maintained at room temperature and mix thoroughly. After incubating the samples for five minutes, transfer the tubes on the magnetic plate and wait another five minutes.Remove and discard the supernatant. Then add 200 microliters of fresh 80%ethanol to the tubes on the magnetic stand. Next, incubate the tubes at room temperature for 30 seconds.After 30 seconds, carefully remove the supernatant and air-dry the beads for five minutes with the lid of the tube open while still on the magnetic stand. Now remove the tubes from the magnet. Elute the target DNA from the beads into 33 microliters of 0.1X Tris-EDTA buffer and subject to thorough mixing.After proper mixing, incubate at room temperature for five minutes. Then transfer the tubes onto the magnetic stand and wait for about two minutes until the solution turns clear. Pipette 30 microliters of the supernatant to two-milliliter tubes for storage.To show the isolation of herbarium DNA, an agarose gel was run with double-stranded DNA isolated from different herbarium specimens. Using 10 milligrams of leaf tissue, almost 3.56 to 2, 610 nanograms of double-stranded DNA isolated was loaded onto the agarose gel. The gel shows degraded herbarium DNA in a smear form in all the lanes.Next, in order to show recovery of high-quality sequencing library from DNA isolated from 10 herbarium specimens, an agarose gel was run. Before loading on the agarose gel, approximately 1.26 to 464 nanograms of the isolated herbarium DNA was subjected to enzymatic shearing and clean up with 90%bead volume. The agarose gel shows recovery of high-quality sequencing library from the DNA of 10 herbarium specimens.Final sequencing libraries show a primary band between 300 to 500 base pairs. Then to show the shotgun sequence of herbarium-derived DNA, a circular plot of TK 686 chloroplast genome was obtained. The sequence shows the total length of the DNA, inverted repeat region, and presence of large and small single-copy regions.Once mastered, this technique can be performed on 24 samples in less than 13 hours with only about eight hours of active hands-on time. After this procedure, other methods, such as sequence capture, can be performed on the resulting libraries to generate nuclear data for phylogenetic studies.

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

Hasta Dokularda metil-bağlayıcı DNA yakalama Dizi

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genetik

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Yüksek verim Protoplast İzolasyonu ve Dönüşüm için Robotik Platformu

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Nucleosome yoğunluk analiz için kitaplıklarına sıralama yerli Kromatin Immunoprecipitation nesil

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Büyük ölçekli gRNA Kütüphane üretim herhangi bir DNA kaynaktan için evrensel bir iletişim kuralı

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: geç genom bölgelerinde çoğaltma tanımlamak için bir yüksek işlem hacmi-dayandırılmış tekniği

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Retrospektif mikroRNA sıralama: Parafin gömülü RNA Formalin sabit örnekler kullanarak Tamamlayıcı DNA Kütüphane hazırlık Protokolü

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Nadiren de olsa algılama hatası düzeltildi DNA ve RNA sıralama

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Ultralow giriş genom Kütüphane hazırlık bir tek Tardigrade örnekten sıralama

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

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Yüksek işlem hacmi DNA ekstraksiyon ve 3dpf zebra balığı larva Fin kırpma tarafından Genotipleme

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Gen düzenleyici dizileri dairesel kromozom Conformation yeni nesil sıralama kullanarak yüksek üretilen iş tanımlaması yakalama (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...