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 consente concentrata, precisa quantificazione metilazione del DNA con elevate capacità di throughput sia il numero di campioni e il numero di obiettivi. Inoltre, questa generazione biblioteca utilizzando tagmentation transposome-mediata richiede meno DNA di ingresso, è più veloce e contiene meno passaggi rispetto tosatura tradizionale e successive tecniche adattatore legatura. Questi miglioramenti rispetto ai metodi attuali consentono precise a livello di base di analisi di metilazione del DNA di qualsiasi target di interesse. Inoltre, BSAS fornisce un nuovo metodo per la conferma delle regioni di interesse (regioni metilazione differenziale (tipo DMRS), isole CpG, regioni di regolamentazione) che sono stati identificati mediante intero genoma bisolfito 22 o catturare 23 approcci. Utilizzando approcci conferma ortogonali e più quantitativamente metodi precisi migliora il rigore analitico degli studi epigenomiche. La profondità elevata di lettura realizzato con BSAS consente per la quantificazione precisa in un intervallo di confidenza stretto, risultazione in quantificazione precisa 16. Inoltre, la possibilità di eseguire molti campioni in un singolo esperimento può aumentare la dimensione del campione per le conferme di metodi intero genoma, che consentirà di aumentare la potenza statistica; una debolezza di molti attuali studi epigenetici. È importante sottolineare che, BSAS fornisce un CpG metilazione quantificazione specifica-base, che consente la generazione di ipotesi verificabili per la ricerca epigenetica. Ipotesi come l&#39;aumento metilazione in un elemento di risposta specifica si tradurrà in una diminuzione legame di un fattore di trascrizione specifico. BSAS, combinati con dati di espressione di mRNA appaiati, fornisce un metodo per ottenere sia la regolazione epigenetica e l&#39;espressione di mRNA in un unico pezzo di tessuto o di una popolazione di cellule. Misure appaiati di regolamentazione e di espressione anche aumentare il rigore scientifico e l&#39;impatto dei risultati. Passaggi critici all&#39;interno del protocollo BSAS comprendono la progettazione di primer, ottimizzazione amplicon, e la generazione di biblioteca /QC. Polarizzazione PCR è introdotto se primer non sono progettati correttamente e amplificano a diverse efficienze 8. L&#39;utilizzo di standard di metilazione, come enzimaticamente generati 100% e 0% citosina standard metilati, può essere utilizzato per determinare il grado, se presente, di polarizzazione metilazione quantificazione, ed è un controllo metodologico appropriato quando quantificare metilazione 16. Allo stesso modo, si deve prestare attenzione quando si ottimizza reazioni PCR per la generazione di un singolo amplicone di alta qualità. Se non amplicone è presente con le linee guida suggerite descritti nel protocollo, passaggi di ottimizzazione includono un numero crescente di ciclo PCR o importo ingresso bsDNA. Se ci sono più prodotti PCR, compreso di primer-dimeri, passaggi di ottimizzazione includono diminuendo quantità di input bsDNA, diminuendo le concentrazioni di ingresso molari PCR Primer, aumento delle temperature di ricottura, o diminuendo il numero di ciclo PCR. Uno o una combinazione di questi procedimenti di ottimizzazione produce risultati sufficienti per generare una singola higamplicon h-qualità. In alternativa, ridisegnando primer è un buon approccio per i set di primer che non producono un singolo amplicone. Per le regioni in cui la riprogettazione non è adatto o possibile, degenerare set di primer, tra cui sia possibile lavorare di citosina e timina all&#39;indirizzo siti CpG in primer avanti e adenina e guanina di di in siti CpG in primer inverso. Tuttavia, questi set di primer devono ottimizzare ulteriormente per garantire l&#39;assenza di pregiudizi PCR è in corso, un processo al di fuori del campo di applicazione di questo protocollo. Librerie di qualità sono estremamente importanti per il successo. Garantire che le misure di controllo di qualità vengono effettuati per determinare le dimensioni, la qualità e la quantità delle librerie prima di sequenziamento. Le reazioni di sequenziamento sono inclini al fallimento con il contributo di librerie di bassa qualità. Bias in metilazione quantificazione può essere mitigato assicurando che le frequenze di metilazione sono presenti in entrambi avanti e retromarcia sequenziamento legge. Inoltre, i punteggi di qualità delle basi allineando a siti CpG dovrebbero essere ≥Q30 per alta confidence in quantificazione. Mentre altri hanno mostrato in precedenza poco o nessun pregiudizio in reazioni tagmentation di bisolfito DNA convertito 19, garantendo i parametri sopra descritti consente la conferma della bassa quantificazione pregiudizi metilazione. BSAS misura attualmente metilazione in siti CpG, però, le future espansioni di questo metodo consentirà misurazioni appaiate del CpG 5-HMC con l&#39;aggiunta di fasi sperimentali distinguendo tra 5-MC e 5-HMC quali l&#39;introduzione di perruthenate potassio prima della conversione bisolfito 24. Ciò aumenterà l&#39;impatto dell&#39;utilità BSAS consentendo metilazione associato, sia dati di espressione 5-mC e 5-HMC, e per determinare espressione genica ruoli normativi di metilazione del DNA. Preparazione biblioteca Transposome-mediata di ampliconi della PCR non comporta approfondito sequenziamento diminuito alle estremità delle regioni amplificate. Pertanto, le regioni di grande interesse dovrebbero essere progettati per risiedere nel mezzo di ampliconi. Comemai, perché BSAS genera sequenziamento leggere profondità di&gt; 1.000 X, la precisione quantitativa di 5-MC misurazioni vicino ai confini delle regioni amplificate rimane elevato.

E &#39;passato più di mezzo secolo da quando il primo rapporto di naturalmente modifiche del DNA in forma di citosina metilazione 1. Citosina metilazione è una base citosina nucleotide modificato in cui il 5 ° di carbonio sull&#39;anello di base ha un gruppo metilico (5-MC), più frequentemente si verificano nel dinucleotide CpG motivo nei genomi dei mammiferi. La presenza funzionale della 5-mC in promotori genici è generalmente associata con la repressione trascrizionale, mentre l&#39;assenza è associato con l&#39;attività trascrizionale 2. Enormi progressi sono stati fatti nella comprensione del ruolo della metilazione del DNA in fase di sviluppo 3, transgeneration propagazione di profili epigenetici 4, cancro patogenesi 5,6, e un certo numero di altri settori di ricerca. Molti di questi progressi sono venuti negli ultimi anni come nuove metodologie sono state sviluppate al profilo di metilazione del DNA 7. Con l&#39;avvento edivulgazione di tecniche di sequenziamento di prossima generazione (NGS) ora ci sono una serie di metodi per profilare la metilazione del DNA di un intero genoma o grandi regioni del genoma 8. Questi approcci aprono la possibilità di studi scoperta che quantificano metilazione del DNA e le differenze di metilazione del DNA. In aggiunta a questi approcci ipotesi che generano, vi è una forte necessità di target / Test di ipotesi DNA metodi metilazione di quantificazione. Pyrosequencing, metilazione specifico PCR, e Sanger sequenziamento diretto del DNA bisolfito convertito sono stati i metodi più utilizzati per l&#39;analisi delle regioni mirati (ad esempio, una regione del promotore di un singolo gene o un&#39;isola CpG) 9-12. Tutti questi metodi si basano sulla conversione bisolfito, una reazione chimica deaminazione dove citosina non metilate del sono convertiti in uracile di, mentre citosina metilati di 13,14 rimangono intatte. Amplificazione PCR di bisulfite-convertiti risultati del DNA in sostituzione di uracile di con timina 15 che consentono la metilazione differenziale leggere come una differenza di base. Mentre molto utile, le limitazioni a questi metodi includono bassa precisione quantitativa, lunghezza lettura breve, e la velocità bassa del campione. Per far fronte a queste limitazioni abbiamo sviluppato un metodo che abbiamo definito bisolfito Amplicon Sequencing (BSAS) 16. L&#39;obiettivo di questo metodo è quello di poter analizzare bersaglio regioni genomiche di interesse in un gran numero di campioni con elevata precisione quantitativa. BSAS utilizza elementi di metodi esistenti (conversione bisolfito e specifiche regione amplificazione PCR) e li combina con semplice costruzione di nuova generazione library (transposome-mediata) 17 e da banco NGS 18 (Figura 1). Questo approccio prevede un metodo di throughput più quantitativa e superiore per esaminare la metilazione citosina in qualsiasi regione di interesse. Ecco a voi laMetodo in dettaglio e descrivere approcci per la risoluzione dei problemi e la qualità controllo del processo 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 si legge correttamente allineato a sequenza di riferimento convertito sarà simile alla Figura 5. Dinucleotides CPG possono chiaramente essere identificati e gli stati di metilazione possono essere stimati osservando le chiamate di base a siti CpG nel mappato legge. Ad esempio, con comandi metilazione, 0% metilazione comporta tutte le letture mappati a siti CpG contenenti T (Figura 5A). 100% dei controlli metilazione comporterà tutta la letture mappato legge a siti contenenti CpG di C (Figura 5B). La quantificazione della frequenza di citosina (C / C + T) in siti CpG produce la frequenza metilazione nel campione originale. Una curva standard generata dal rappresentante interi controlli genoma metilazione (n = 3 / rapporto metilazione) mostra linearità metilazione quantificazione nonché precisione quantificazione a ciascun rapporto metilazione (Figura 6). Come esempio di questo metodo in totale, RNA e DNA sono stati co-iso<html> lata di topo cervelletto e retina (n = 4 / group). Espressione Rhodopsin, selettivamente espresso in tessuto retinico, è stata misurata utilizzando qPCR. Expression è stata rilevata in retina (Figura 7A). Utilizzando BSAS, i livelli di metilazione CpG sono stati quantificati nella regione del promotore rodopsina. Livelli di metilazione cumulativi tutta la regione promotore erano&gt; 80% nel cervelletto rispetto al &lt;15% nella retina (p &lt;0,001, t-test parametrico) (Figura 7B). BsAs benefici metilazione di quantificazione di site-specific metilazione quantificazione, e livelli di metilazione possono essere confrontati su base CpG-specifica attraverso una determinata regione genomica. Livelli di metilazione CpG in tutto il promotore rodopsina erano significativamente più alti nei campioni cerebellari rispetto ai campioni di retina (p &lt;0.001, t-test parametrico in ciascun sito CpG) (Figura 7c). 1highres.jpg &quot;/&gt; Figura 1:. Schema del metodo BSAS In questo metodo, il DNA genomico è bisolfito convertito modificare cytosines unmethylation a uracils. Successivamente, durante la PCR questi uracils vengono modificati a timina. Amplificazione PCR è diretto contro le regioni di interesse e altamente arricchisce solo per queste sequenze. I risultanti ampliconi della PCR sono fatti in librerie dual-indicizzati attraverso un semplice processo tagmentation. Successivamente le librerie sono in sequenza su un banco di nuova generazione sequencer e il sequenziamento letture sono mappati in silico convertito sequenza di riferimento e cento metilazione della citosina di è determinata in modo specifico-base. <img alt="Figura 2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> Figura 2:. In silico conversione bisolfito di regione di interesse Tutte le regioni di interesse da ogni riferimento genomico può essere Selected per la conversione in silico bisolfito. Cytosines non-CpG sono sostituiti con timina nella sequenza di riferimento. Cytosines CpG restano cytosines del bisolfito convertiti riferimento. <img alt="Figura 3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> Figura 3:. Primer placement Primer bisolfito PCR sono progettati contro una in silico bisolfito convertito sequenza di riferimento. Primer devono essere progettati in modo che non si sovrappongano dinucleotidi CG o progettati adiacente dinucleotidi CG. <img alt="Figura 4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> Figura 4: Esempi di ampliconi alti e poveri PCR qualità e le librerie di sequenziamento elettroferogramma tracce rappresentative e gel spettacolo (A) amplicone ideale per la generazione biblioteca transposome mediata con una sola alta.Prodotto concentrazione. (B) Biblioteca generata da questo amplicon mostra elevata concentrazione e la distribuzione, anche le dimensioni. (C) scarso ampliconi della PCR qualità possono essere bassi in termini di dimensioni, la concentrazione, e contengono più prodotti e / o dimeri di primer. Questi ampliconi di scarsa qualità porterà a (D) fallito generazione biblioteca a bassa concentrazione e la distribuzione bassa dimensione. <img alt="Figura 5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> Figura 5: Esempi di uscite di sequenziamento di controlli metilazione. (A) In silico convertito sequenziamento di riferimento con i siti CpG evidenziata in rosso. 0% di controllo metilazione mappato legge alla regione mostrando timina selezionati (avidità evidenziare) mappatura a siti CpG. Di controllo (B) 100% metilazione mappato legge mostra (blu highlight) mappatura di citosina in siti CpG. Figura 6:. Quantificazione metilazione in una serie di controlli di metilazione dell&#39;intero genoma controlli metilazione mescolato a rapporti di metilazione (n = 3 / ratio) da 0% a 100% sono stati quantificati con BSAS. Tracciamento quantificazione del previsto contro quantificata dimostra linearità del controllo metilazione quantificazione. Attraverso tutti i controlli, si è ad alta precisione in metilazione quantificazione. <img alt="Figura 7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> Figura 7: Esempio di abbinato metilazione del DNA ed espressione genica analisi da campioni di tessuto. (A) Rhodopsin espressione di mRNA relativa dal topo cerebellare e tessuto retinico. (B) Rhodopsin metilazione media promotore quantificato da BSAS tra cerebellare e il DNA della retina (*** p &lt;0.001, parametrico t-test). &lt;strong&gt; livelli di metilazione C) sito-specifica CpG tutto promotore rodopsina (-244 a +6, rispetto al sito di inizio della trascrizione [TSS]) nel topo cervelletto e DNA retina.

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.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

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

Research

JoVE Journal

Biology

36.9K Views.  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.

Video: 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.

Determinazione della metilazione del DNA di geni impressa Arabidopsis Dell&#39;endosperma

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

Ricerca

Biologia

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.

Metilazione del DNA: Modifica bisolfito e analisi

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.

Microscopia a fluorescenza-Screening e sequenziamento di prossima generazione: Strumenti utili per l&#39;identificazione di geni coinvolti in organelli Integrity

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.

Sequenziamento di prossima generazione del 16S RNA ribosomiale Gene ampliconi

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 ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

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.

Amplificazione, sequenziamento di prossima generazione, e DNA genomico mappatura dei siti di integrazione retrovirali

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.

Interactoma-Seq: Un protocollo per la costruzione della libreria Domainome, la convalida e la selezione di Phage Display e sequenziamento di nuova generazione

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.

Tick Microbiome caratterizzazione ordinando Amplicon del rRNA 16S di prossima generazione

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.

Analisi microbiota utilizzando PCR in due fasi e sequenziamento genico rRNA 16S di nuova generazione

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 protocollo di estrazione del DNA delle zanzare a base di perline magnetiche per il sequenziamento di nuova generazione

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