The authors thank Colleen Van Kirk for mouse retina and cerebellum samples, and Peter Gregory for figure generation. The authors also thank Dr. Allison Gillaspy and the Laboratory for Molecular Biology and Cytometry Research core for access to the MiSeq. This work was supported by NIH grants DA029405, EY021716, and AG026607 and the Donald W. Reynolds Foundation.

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The authors have nothing to disclose.

BSAS permet de concentré, la quantification précise de méthylation d&#39;ADN avec des capacités de débit élevées à la fois dans le nombre d&#39;échantillons, le nombre de cibles. En outre, cette génération bibliothèque à l&#39;aide de tagmentation transposome médiation nécessite l&#39;ADN d&#39;entrée moins, est plus rapide et contient moins d&#39;étapes que cisaillement traditionnelle et les techniques de ligature adaptateur ultérieures. Ces améliorations par rapport aux méthodes existantes permettent précise le niveau base de l&#39;analyse de méthylation d&#39;ADN de toute cible d&#39;intérêt. En outre, BSAS fournit une nouvelle méthode de confirmation de régions d&#39;intérêt (régions de méthylation de manière différentielle (DMR), îlots CpG, régions réglementaires) qui ont été identifiés à l&#39;aide de génomes entiers bisulfite 22 ou capturer 23 approches. En utilisant des approches de confirmation orthogonales et plus quantitativement méthodes précises améliore la rigueur analytique des études épigénomiques. La profondeur de lecture élevé atteint avec BSAS permet une quantification précise dans un intervalle de confiance étroit, rectsconsulting dans la quantification précise 16. En outre, la capacité d&#39;exécuter de nombreux échantillons en une seule expérience peut augmenter la taille de l&#39;échantillon pour les confirmations de méthodes du génome entier, ce qui augmentera la puissance statistique; une faiblesse de nombreuses études épigénétiques actuelles. Surtout, BSAS fournit une quantification spécifique de méthylation CpG-base, ce qui permet la génération d&#39;hypothèses vérifiables pour la recherche en épigénétique. Les hypothèses comme l&#39;augmentation de la méthylation à un élément de réponse spécifique provoque une diminution de la liaison d&#39;un facteur de transcription spécifique. BSAS, combinées avec les données d&#39;expression d&#39;ARNm couplés, fournit un procédé d&#39;obtention à la fois la régulation épigénétique et l&#39;expression de l&#39;ARNm au sein d&#39;une seule pièce de tissu ou population de cellules. Mesures paires de régulation et l&#39;expression augmentent également la rigueur scientifique et l&#39;impact des résultats. Les étapes critiques au sein du protocole BSAS comprennent la conception primaire, l&#39;optimisation de l&#39;amplicon, et la génération de bibliothèque /QC. Biais PCR est introduit si amorces sont pas bien conçus et amplifient l&#39;efficacité à différents 8. L&#39;utilisation de normes de méthylation, comme 100% et 0% cytosine méthylés normes générés par voie enzymatique, peut être utilisé pour déterminer le degré, le cas échéant, de partialité méthylation de quantification, et est un contrôle méthodologique appropriée lors de la quantification méthylation 16. De même, il faut prendre soin lors de l&#39;optimisation des réactions de PCR pour générer un seul amplicon de haute qualité. Si aucun amplicon est présent en utilisant les lignes directrices proposées décrites dans le protocole, des mesures d&#39;optimisation comprennent l&#39;augmentation du nombre de cycles PCR ou le montant d&#39;entrée bsDNA. Se il ya des produits de PCR multiples, y compris des dimères d&#39;amorces, des mesures d&#39;optimisation comprennent quantité décroissante d&#39;entrée bsDNA, diminuant les concentrations d&#39;entrée molaires d&#39;amorces PCR, la hausse des températures de recuit, ou en diminuant le nombre de cycles PCR. L&#39;un ou une combinaison de ces procédures d&#39;optimisation donne des résultats suffisants pour générer une seule higamplicon h-qualité. Alternativement, la refonte amorces est une bonne approche pour jeux d&#39;amorces qui ne produisent pas un seul amplicon. Dans les régions où la refonte ne est pas approprié ou possible, dégénérer jeux d&#39;amorces dont les deux peuvent travailler de la cytosine et la thymine de sites CpG dans amorces sens et de l&#39;adénine et la guanine de sur les sites CpG dans amorces inverses. Cependant, ces jeux d&#39;amorces doivent poursuite de l&#39;optimisation pour se assurer qu&#39;aucun biais PCR est en cours, un processus en dehors du champ d&#39;application de ce protocole. bibliothèques de qualité sont extrêmement important pour le succès. Assurez-vous que QC mesures sont effectuées pour déterminer la taille, la qualité et la quantité des bibliothèques avant séquençage. Les réactions de séquençage sont sujettes à l&#39;échec avec la participation des bibliothèques de faible qualité. Biais dans la méthylation quantification peut être atténué en veillant à ce que les fréquences de méthylation sont présents à la fois avant et arrière lit séquençage. De plus, les scores de qualité de l&#39;alignement des bases de sites CpG devraient être ≥Q30 pour la haute confidence dans la quantification. Alors que d&#39;autres ont montré précédemment peu ou pas de biais dans les réactions de tagmentation de bisulfite ADN converti 19, assurant que les mesures décrites ci-dessus permet de confirmation de faible quantification biais de méthylation. BSAS mesure actuellement méthylation des sites CpG, cependant, de futures extensions de cette méthode permettra mesures paires de CpG 5-HMC avec l&#39;ajout d&#39;étapes expérimentales distinction entre 5-mC et 5-HMC telles que l&#39;introduction perruthénate de potassium avant la conversion au bisulfite 24. Cela permettra d&#39;accroître l&#39;impact de l&#39;utilité BSAS en permettant pour la méthylation jumelé, deux 5-mC et 5-HMC, et des données d&#39;expression afin de déterminer les rôles régulation de l&#39;expression des gènes de méthylation de l&#39;ADN. Préparation de bibliothèque Transposome médiée par des amplicons PCR ne entraîne une diminution de la profondeur de séquençage aux extrémités des régions amplifiées. Par conséquent, les régions d&#39;un grand intérêt devraient être conçus pour résider dans le milieu de amplicons. Commentjamais, parce que BSAS génère séquençage lire profondeurs de&gt; 1000 X, la précision quantitative de 5-mC mesures près des extrémités des régions amplifiées reste élevé.

Il a été plus d&#39;un demi-siècle, depuis le premier rapport de modifications de l&#39;ADN d&#39;origine naturelle sous forme de méthylation de cytosine 1. La méthylation des cytosines est une base cytosine nucleotide modifié où la 5 ème carbone sur l&#39;anneau de base a un groupe méthyle (5-mC), se produisant le plus fréquemment dans le dinucléotide CpG motif dans les génomes de mammifères. La présence fonctionnelle de 5-mC dans les promoteurs de gènes est généralement associée à la répression transcriptionnelle, tandis que l&#39;absence est associée à une activité de transcription 2. D&#39;énormes progrès ont été réalisés dans notre compréhension du rôle de la méthylation de l&#39;ADN dans le développement 3, transgénérationnelle propagation de profils épigénétiques 4, la pathogenèse du cancer 5,6, et un certain nombre d&#39;autres domaines de recherche. Beaucoup de ces progrès sont venus au cours des dernières années que de nouvelles méthodes ont été développées pour le profil de méthylation de l&#39;ADN 7. Avec l&#39;avènement etpopularisation de séquençage de prochaine génération (NGS) techniques il ya maintenant un certain nombre de méthodes pour le profil méthylation de l&#39;ADN dans un génome entier ou de grandes régions d&#39;un génome huit. Ces approches ouvrent la possibilité d&#39;études de découverte qui permettent de quantifier les modèles et les différences de méthylation de l&#39;ADN méthylation ADN. En plus de ces approches hypothèse génératrices, il existe un besoin important pour cible / ADN hypothèse de test méthodes méthylation de quantification. Pyroséquençage, PCR spécifique de la méthylation et séquençage Sanger directe de l&#39;ADN converti au bisulfite ont été les méthodes les plus utilisées pour l&#39;analyse des régions ciblées (ce est à dire, une région promotrice d&#39;un seul gène ou d&#39;un îlot CpG) 9-12. Toutes ces méthodes reposent sur ​​la conversion au bisulfite, une réaction de désamination chimique où la cytosine non méthylées sont converties en de de l&#39;uracile, la cytosine méthylés tandis que de 13,14 restent intacts. L&#39;amplification par PCR de bisulfite-Résultats d&#39;ADN converties en uracile de remplacement de la thymine avec 15 permettant la méthylation différentielle à lire comme une différence de base. Bien que très utile, les limites de ces méthodes comprennent faible précision quantitatives, faible longueur de lecture, faible capacité, et d&#39;échantillon. Pour répondre à ces limitations nous avons développé une méthode que nous avons appelé bisulfite Amplicon séquençage (BSAS) 16. L&#39;objectif de cette méthode est d&#39;être capable d&#39;analyser des régions génomiques cibles d&#39;intérêt dans un grand nombre d&#39;échantillons avec une précision quantitative élevé. BSAS utilise des éléments de méthodes existantes (conversion au bisulfite et l&#39;amplification de la PCR spécifique à la région) et les combine avec une simple construction de la prochaine génération bibliothèque (transposome médiation) 17 et paillasse NGS 18 (Figure 1). Cette approche fournit un procédé de débit quantitatif plus élevé et à examiner la méthylation de la cytosine dans ne importe quelle région d&#39;intérêt. Nous présentons ici laméthode en détail et décrire des approches pour le dépannage et vérification de la qualité du processus BSAS.

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Name</td>
			<td style="width:255px;">Company</td>
			<td style="width:97px;">Cat #</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent 2100 Bioanlyzer</td>
			<td>Agilent</td>
			<td>G29393AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent RNA 6000 Nano kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent High Sensitivity DNA kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9200</td>
			<td>Amersham/GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt AMPure XP - PCR Purification</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>5 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">twin.tech PCR plate 96&#160;</td>
			<td>Eppendorf</td>
			<td>951020362</td>
			<td>semi-skirted</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Film</td>
			<td>Eppendorf</td>
			<td>0030127838</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Foil</td>
			<td>Eppendorf</td>
			<td>0030127854</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat Sealer</td>
			<td>Eppendorf</td>
			<td>5390000.024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1-10ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-200ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.045</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-1000ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MixMate&#160;</td>
			<td>Eppendorf</td>
			<td>5353000.014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>0030121.023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td>5424000.010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0 mL Microcentrifuge Tube</td>
			<td>Eppendorf</td>
			<td>022363352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>24 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td>24 indexes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Desktop Sequencer</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Reagent Kit v2</td>
			<td>Illumina</td>
			<td>MS-102-2002</td>
			<td>300cycle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KAPA SYBR FAST Universal qPCR kit</td>
			<td>KAPA Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT PicoGreen dsDNA Assay kit</td>
			<td>Life Technologies</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT RiboGreen RNA Assay kit</td>
			<td>Life Technologies</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ProFlex 96-well PCR system</td>
			<td>Life Technologies</td>
			<td>4484075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex 6% TBE DNA gel</td>
			<td>Life Technologies</td>
			<td>EC6261BOX</td>
			<td>10-well</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex TBE Running Buffer</td>
			<td>Life Technologies</td>
			<td>LC6675</td>
			<td>5X (1 L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex Hi-Density TBE Sample Buffer</td>
			<td>Life Technologies</td>
			<td>LC6678</td>
			<td>5X (10 mL)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Kb Plus DNA Ladder</td>
			<td>Life Technologies</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure 10mg/mL Ethidium Bromide</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7900HT Fast Real-Time PCR system</td>
			<td>Life Technologies</td>
			<td>4329001</td>
			<td>384well block</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DynaMag-96 Side Skirted</td>
			<td>Life Technologies</td>
			<td>12027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SpectroMax M2 Multi-Mode Microplate Reader</td>
			<td>Molecular Devices/VWR</td>
			<td>89429-532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Beads</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td>5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLC Genomics Workbench</td>
			<td>Qiagen</td>
			<td></td>
			<td>Software for methylation pipeline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAQuick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TissueLyser II</td>
			<td>Retsch/Qiagen</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-Free Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TRIS hydrochloride</td>
			<td>Sigma</td>
			<td>PHG0002-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween-20</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">384-well Full-Skirted Plate, Standard</td>
			<td>Thermo Scientific</td>
			<td>AB-1384</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absolute qPCR Seal</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EZ DNA Methylation-Lightning kit</td>
			<td>Zymo Research</td>
			<td>D5030</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZymoTaq DNA Polymerase</td>
			<td>Zymo Research</td>
			<td>E2001</td>
			<td>50 rxn</td>
		</tr>
	</tbody>
</table>

targeted dna methylation analysis by next-generation sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

BSAS lectures correctement aligné à la séquence de référence convertie ressemblera Figure 5. Dinucléotides CpG peuvent être clairement identifiés et les états de méthylation peuvent être estimées en observant les appels de base sur les sites CpG dans le lit mappé. Par exemple, avec des commandes de méthylation, 0% méthylation se traduira par toutes les lectures mises en correspondance avec les sites CpG contenant (figure 5A) de T. 100% des contrôles de méthylation se traduiront par toutes les lectures mappé à lit contenant des sites CpG (figure 5B) de C. La quantification de la fréquence de la cytosine (C / C + T) à des sites CpG donne la fréquence de méthylation dans l&#39;échantillon d&#39;origine. Une courbe étalon générée à partir de commandes représentant de méthylation du génome entier (n = 3 / taux de méthylation) montre la linéarité de la méthylation de quantification ainsi que la précision de quantification à chaque taux de méthylation (figure 6). A titre d&#39;exemple de ce procédé, au total, de l&#39;ARN et de l&#39;ADN ont été co-iso<html> culé des cervelet de souris et de la rétine (n = 4 / groupe). Expression rhodopsine, sélectivement exprimée dans les tissus de la rétine, a été mesurée en utilisant qPCR. L&#39;expression a été détectée seulement dans la rétine (figure 7A). Utilisation BSAS, les niveaux de méthylation de CpG ont été quantifiés dans la région de promoteur de la rhodopsine. Les niveaux de méthylation cumulatifs à travers la région de promoteur était&gt; 80% dans le cervelet par rapport à &lt;15% dans la rétine (p &lt;0,001, test t paramétrique) (Figure 7B). BSAS quantification avantages de méthylation spécifique du site de méthylation ainsi la quantification des niveaux de méthylation peuvent être comparés sur une base spécifique de CpG à travers une région génomique donnée. CpG niveaux de méthylation de tout le promoteur de la rhodopsine étaient significativement plus élevés dans les échantillons cérébelleux échantillons par rapport à la rétine (p &lt;0,001, test t paramétrique sur chaque site CpG) (Figure 7c). 1highres.jpg &quot;/&gt; Figure 1:. Schéma de la méthode BSAS Dans ce procédé, l&#39;ADN génomique est converti au bisulfite de modifier les cytosines de unmethylation à uraciles. Par la suite, au cours de ces PCR uraciles sont modifiés pour thymines. Amplification par PCR est dirigé contre les régions d&#39;intérêt et très enrichit pour seulement ces séquences. Les amplicons PCR résultants sont transformés en bibliothèques double indexées par un processus de tagmentation simple. Ensuite, les bibliothèques sont séquencés sur un séquenceur paillasse de la prochaine génération et le séquençage lectures sont mappés sur la converti in silico séquence de référence et pour cent de méthylation de la cytosine est déterminée d&#39;une manière spécifique à la base. <img alt="Figure 2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> Figure 2:. Dans silico conversion au bisulfite de région d&#39;intérêt Toute région d&#39;intérêt de toute référence génomique peut être sélecTed in silico pour conversion au bisulfite. Cytosines non-CpG sont remplacés par de la thymine dans la séquence de référence. Cytosines CpG restent cytosines converties au bisulfite dans la référence. <img alt="Figure 3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> Figure 3:. Amorces placement Primer bisulfite PCR sont conçus contre une in silico bisulfite converti séquence de référence. Amorces doivent être conçus de sorte qu&#39;ils ne se chevauchent pas dinucléotides CG ou conçus à côté de dinucléotides CG. <img alt="Figure 4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> Figure 4: Exemples de amplicons de PCR de haute qualité et pauvres et les bibliothèques de séquençage traces de électrophérogramme représentatifs et son émission de gel (A) de amplicon idéal pour la génération de la bibliothèque de transposome médiation avec un seul haut.produit de concentration. (B) Bibliothèque généré à partir de ce amplicon montre une forte concentration et même la distribution de taille. (C) amplicons de PCR de mauvaise qualité peuvent être faible en taille, la concentration, et contient de multiples produits et / ou de dimères d&#39;amorces. Ces amplicons de mauvaise qualité conduiront à (D) a échoué génération bibliothèque faible concentration et la distribution de faible taille. <img alt="Figure 5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> Figure 5: Exemples de résultats de séquençage de contrôles de méthylation. (A) Dans silico converti séquençage de référence avec des sites CpG sur fond rouge. 0% de contrôle de méthylation mappé lit de la région indiquant thymines sélectionnés (la cupidité de surbrillance) de cartographie des sites CpG. Commande (B) 100% de méthylation mappé lit montrer (surbrillance bleue) la cartographie de cytosine sur les sites CpG. Figure 6:. Méthylation quantification travers une série de contrôles de méthylation contrôles de méthylation du génome tout mélangé à des taux de méthylation (n = 3 / Ratio) de 0% à 100% ont été quantifiés en utilisant BSAS. Tracé de la quantification de prévu contre quantifiée démontre linéarité du contrôle de méthylation quantification. Dans tous les contrôles, il y avait une grande précision dans la méthylation de quantification. <img alt="Figure 7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> Figure 7: Exemple de paires méthylation de l&#39;ADN et l&#39;expression du gène à partir de l&#39;analyse des échantillons de tissus. (A) Rhodopsin relatif d&#39;expression d&#39;ARNm à partir du cervelet de la souris et du tissu rétinien. (B) la méthylation du promoteur Rhodopsin moyenne quantifiée par BSAS entre cérébelleux et de l&#39;ADN de la rétine (*** p &lt;0,001, test t paramétrique). &lt;forts niveaux de méthylation&gt; C) spécifique de CpG à travers le site promoteur de la rhodopsine (-244 à 6, par rapport au site d&#39;initiation de transcription [TSS]) dans le cervelet de souris et de l&#39;ADN rétine.

Watch this Scientific Journal Video about Targeted DNA Methylation Analysis by Next-generation Sequencing at JoVE.com

Targeted DNA Methylation Analysis by Next-generation Sequencing

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Ciblé méthylation de l&#39;ADN Analyse par séquençage de nouvelle génération

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

targeted-dna-methylation-analysis-by-next-generation-sequencing

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Biology

36.9K vues.  University of Oklahoma College of Medicine. Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Vidéo: Targeted DNA Methylation Analysis by Next-generation Sequencing

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Détermination de méthylation de l&#39;ADN des gènes soumis à empreinte dans les Arabidopsis Endosperme

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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Biologie

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Méthylation de l&#39;ADN: bisulfite de modification et d&#39;analyse

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

Fluorescence microscopie dépistage et séquençage de prochaine génération: des outils utiles pour l&#39;identification des gènes impliqués dans des organites intégrité

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

La nouvelle génération de séquençage de l&#39;ARN ribosomal 16S gènes amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced réduit Représentation bisulfite séquençage pour l&#39;évaluation de méthylation de l&#39;ADN à la résolution de paires de base

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Séquençage de nouvelle génération, et l&#39;ADN génomique Cartographie des rétrovirales Sites d&#39;intégration

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome-Seq : Un protocole pour la Construction de la bibliothèque de Domainome, de Validation et de sélection par Phage Display et de prochaine génération séquençage

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Tique Microbiome caractérisation par génération 16 s rRNA Amplicon séquençage

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Analyse du microbiote à l'aide de PCR en deux étapes et de la prochaine génération 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Un protocole d’extraction de l’ADN des moustiques à base de perles magnétiques pour le séquençage de nouvelle génération

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...