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 샘플 및 대상의 수 두 수의 높은 처리량 기능에 초점을 맞춘, 정확한 DNA 메틸화 정량 수 있습니다. 또한, transposome 매개 tagmentation을 사용하여이 라이브러리 세대는 빠르고, 적은 입력 DNA입니다 필요로하며, 기존의 전단 및 후속 어댑터 결찰 기술보다 더 적은 단계가 포함되어 있습니다. 기존의 방법에 비해 이러한 개선은 관심의 대상의 정확한 기본 수준 DNA 메틸화 분석 할 수 있습니다. 또한, BSAS는 전체 게놈 중아 (22) 또는 캡처 (23) 접근 방식을 사용하여 식별 된 관심의 영역 (차별적 메틸화 지역 (DMRS)의 CpG 섬, 규제 지역)의 확인을위한 새로운 방법을 제공한다. 직교 확인 방법 등을 정량적으로 정확한 방법을 사용하여 에피 지노믹스 연구의 치밀한 분석을 향상시킨다. BSAS 달성 높은 읽기 깊이는 다시 좁은 신뢰 구간의 정확한 정량이 가능정확한 정량 16 sulting. 또한, 하나의 실험에서 많은 샘플을 실행할 수있는 능력은 통계적 전력을 증가 전체 게놈의 확정 방법에 대한 샘플 크기를 증가시킬 수있다; 많은 현재의 후생 유전 학적 연구의 약점. 중요한 것은, BSAS은 후생 유전 학적 연구에 대한 검증 가능한 가설의 생성을 허용베이스 고유의 CpG 메틸화 정량을 제공합니다. 이러한 특정 응답 요소의 증가 메틸화와 같은 가설이 감소 특정 전사 인자의 결합을 발생합니다. 한 쌍의 mRNA 발현 데이터와 결합 BSAS은 조직 또는 세포 집단의 단일 조각 내에 후생 유전 학적 규제 mRNA 발현 양을 얻기위한 방법을 제공한다. 규제와 표현의 짝 측정은 또한 과학적인 엄격함과 결과의 영향을 증가시킨다. BSAS 프로토콜 내에서 중요한 단계는 프라이머 디자인, 앰플 리콘 최적화 및 라이브러리 생성 / 포함QC. 프라이머가 적절하게 설계 효율이 다른 8로 증폭하지 않으면 PCR 바이어스가 도입된다. 이러한 효소 적으로 생성 된 100 % 및 0 % 시토신 메틸화 표준 같은 메틸화 표준의 사용은 어떤 경우 메틸화 정량 바이어스, 정도를 결정하는데 사용하고, 메틸화 (16)을 정량 할 때 적절한 방법론 제어하다 할 수있다. 단일 고품질의 증폭 물을 생성하기위한 PCR 반응을 최적화 할 때 마찬가지로주의를 기울여야한다. 앰플 리콘은 어떠한 프로토콜에서 상세히 제시된 지침을 이용하여 존재하지 않는 경우, 최적화 단계가 증가하는 PCR 사이클 번호 또는 bsDNA 입력량을 포함한다. 프라이머 이량 체 등 다양한 PCR 제품들이 존재하는 경우, 최적화 단계는, bsDNA 입력량 감소 PCR 프라이머 몰 입력 농도 저하, 소둔 온도의 증가, 또는 PCR 싸이클 횟수 감소 등. 이러한 최적화 절차 중 하나 또는 조합이 단일 HIG를 생성하기에 충분한 결과를 얻을H-품질 증폭 물. 또한, 프라이머를 재 설계하는 것은 하나의 증폭을 생성하지 않는 프라이머 세트를위한 좋은 방법입니다. 재 설계에 적합 또는 수 없습니다 그 지역의 경우 두 역 프라이머에서의 CpG 사이트에서의 CpG 앞으로 프라이머의 사이트와 아데닌의 구아닌의에서 시토신의 티민의이 작동을 포함 프라이머 세트를 퇴화. 그러나, 이들 프라이머 세트는 바이어스가 발생하는 어떠한 PCR,이 프로토콜의 범위를 벗어난 프로세스 없도록 위해 추가 최적화를 필요로한다. 품질 라이브러리는 성공을 위해 매우 중요합니다. 품질 관리 조치를 시퀀싱하기 전에 라이브러리의 크기, 질과 양을 결정하기 위해 수행되어 있는지 확인합니다. 시퀀싱 반응은 저품질 라이브러리의 입력에 실패하는 경향이있다. 메틸화 정량의 바이어스는 메틸화 주파수가 앞으로 모두에 존재하고 시퀀싱 읽기 역하도록함으로써 완화 될 수있다. 또한,의 CpG 사이트에 정렬 기지의 품질 평가 점수가 높은 협력을위한 ≥Q30해야정량에 nfidence. 다른 사람은 위에 설명 된 기준을 보장 중아 변환 된 DNA (19)의 tagmentation 반응에서 어떤 편견 이전에 작은 표시하고 있지만 낮은 바이어스 메틸화 정량의 확인을 할 수 있습니다. BSAS 현재 그러나,이 방법의 미래 확장은 바이 설 파이트 변환 24 전에 칼륨 퍼 루테 네이트를 도입으로하는 5- MC 및 5- HMC 구별 실험 단계에 첨가 된 CpG -5- HMC의 페어링 측정을 허용 소비재 부위에서 메틸화를 측정한다. 이것은 DNA 메틸화 유전자 발현 조절의 역할을 결정하기 위해, 한 쌍의 메틸화시킴으로써 5-MC 및 5- HMC 및 발현 데이터를 모두 BSAS 유틸리티의 영향을 증가시킬 것이다. PCR 증폭 산물의 Transposome 매개 라이브러리 제조 증폭 영역의 단부에서 감소 된 깊이 순서화 될 않는다. 따라서, 높은 관심의 영역이 증폭 산물의 중간에 위치하도록 설계되어야한다. 방법BSAS가 생성하기 때문에 지금까지, 시퀀싱&gt; 1000 X, 증폭 된 영역의 끝 근처 측정이 여전히 높은 5-MC의 양적 정도의 깊이를 읽어보십시오.

그것은 자연스럽게 시토신 메틸화 (1)의 형태로 DNA의 변형 발생의 첫 번째 보고서 이후 반세기에 걸쳐있다. 시토신 메틸화베이스 링에 5 번째 탄소 메틸기 (5-MC)는, 가장 빈번하게 포유 동물 게놈에서의 CpG 디 뉴클레오티드 모티브에서 발생 갖는 변성 시토신 뉴클레오티드 염기이다. 부재가 전사 활성과 연관이있는 동안 유전자 프로모터의 5-MC의 존재는 일반적으로 작용 성, 전사 억제와 관련된다. 엄청난 진보 발전 3의 DNA 메틸화, 후생 유전 학적 프로파일 4, 5,6 암 발병 기전 및 다른 연구 분야의 수의 transgeneration 전파의 역할에 대한 이해 만들어왔다. 신규 방법론은 DNA 메틸화 7 프로파일 개발되었다 이러한 많은 진보는 지난 몇 년 동안왔다. 출현 및차세대 시퀀싱 (NGS) 기술의 대중화는 이제 전체 게놈 또는 게놈 (8)의 큰 영역에 걸쳐 DNA 메틸화를 프로파일 링하는 방법은 여러 가지가있을 수 있습니다. 이러한 접근은 DNA 메틸화 패턴 및 DNA 메틸화의 차이를 정량화 연구의 발견 가능성을 연다. 이러한 가설 발생 방법에 더하여, 타겟 / 가설 테스트 DNA 메틸화 정량 방법에 대한 상당한 필요성이 존재한다. pyrosequencing 기법, 메틸화 특이 PCR, 바이 설 파이트 변환 된 DNA와 직접 생거 시퀀싱은 대상 영역의 분석을 위해 가장 많이 사용되는 방법이 있었다 (예를 들어, 하나의 유전자의 프로모터 영역 또는 CpG 아일랜드) 9-12. 메틸화 된 시토신의 그대로 13, 14을 유지하면서 이러한 모든 방법, 중아 변환, 메틸화 된 시토신의이 우라실의로 변환 화학 탈 아미 노화 반응에 의존하고있다. bisulfite-의 PCR 증폭차동 메틸화 염기 차이로서 판독 할 수 티민 15 우라실의 여분의 DNA에 변환 결과. 매우 유용하지만, 이러한 방법에 제한이 낮은 양적 정확성, 짧은 읽기 길이, 낮은 시료 처리를 포함한다. 이러한 한계를 해결하기 위해 우리는 우리가 (BSAS) 16 바이 설 파이트의 증폭 시퀀싱이라고 한 방법을 개발했다. 이 방법의 목적은 높은 정밀도로 정량 많은 수의 샘플에 대한 관심의 대상 게놈 영역을 분석 할 수 있어야한다. BSAS은 기존의 방법의 요소 (중아 변환 및 지역별 PCR 증폭)를 사용하고 간단한 차세대 라이브러리 건설 (transposome 매개) (17) 및 벤치 탑 NGS (18) (그림 1)로 결합되어 있습니다. 이 방법은 관심의 영역에서 시토신 메틸화를 검사 할 수있는 정량적 및 높은 처리량 방법을 제공한다. 여기에 우리가 제시세부 방법 및 문제 해결과 품질이 BSAS 프로세스를 확인하는 방법을 설명합니다.

<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Agilent 2100 Bioanlyzer</td>
			<td>Agilent</td>
			<td>G29393AA</td>
			<td></td>
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			<td height="20" style="height:20px;">Agilent RNA 6000 Nano kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
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			<td height="20" style="height:20px;">Agilent High Sensitivity DNA kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9200</td>
			<td>Amersham/GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Agencourt AMPure XP - PCR Purification</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>5 mL</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio Rad</td>
			<td>164-5050</td>
			<td></td>
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			<td height="20" style="height:20px;">twin.tech PCR plate 96&#160;</td>
			<td>Eppendorf</td>
			<td>951020362</td>
			<td>semi-skirted</td>
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			<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>
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		<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 제대로도 5를 닮은 것 변환 된 레퍼런스 시퀀스에 정렬 읽는다. CpG 디 뉴클레오티드는 명확히 식별 할 수 있고, 메틸화 상태는 읽기에 매핑 된 CpG 부위에서베이스 호출을 관찰함으로써 추정 될 수있다. 예를 들어, 메틸화 컨트롤, 0 %는 모든 메틸화가 T의 (도 5a)를 함유 된 CpG 부위에 매핑 판독 될 것이다. 100 % 메틸화 컨트롤 매핑 읽기 모두가 C의 (그림 5B)를 포함 된 CpG 사이트에 대한 읽기가 발생합니다. 된 CpG 부위에서 주파수 시토신 (C / C + T)의 정량은 원래 샘플 메틸화 주파수를 산출한다. 전체 게놈의 메틸화 컨트롤 (N = 3 / 메틸화 비율)에서 발생하는 대표적인 표준 곡선은 각 메틸화 비율 (그림 6)에서 정량의 메틸화 정량의 선형성뿐만 아니라 정밀도를 보여줍니다. 총이 방법의 예로서, RNA 및 DNA는 공동 ISO이었다<html> 마우스 소뇌와 망막 (N = 4 / 그룹)에서 lated. 선택적 망막 조직에서 발현 로돕신 발현, qPCR을 사용하여 측정 하였다. 표현은 망막 (그림 7A)에서 검출되었다. BSAS를 사용하여, 메틸화 된 CpG 수준은 로돕신의 프로모터 영역에서 정량화 하였다. 프로모터 영역에 걸쳐 누적 메틸화 수준은 망막 (p &lt;0.001, 파라 메트릭 테스트 t) (도 7B)에서 &lt;15 %에 비해 소뇌에서&gt; 80 %이었다. 부위 특이 적 메틸화 정량로부터 BSAS 메틸화 정량 이점 및 메틸화 수준은 특정 지역 전체 게놈의 CpG 관련 기초 비교 될 수있다. 망막 샘플 (p &lt;0.001, 각 사이트에서의 CpG 파라 메트릭 테스트 t) (도 7C)에 비해 로돕신 프로모터 걸쳐 메틸화 된 CpG 소뇌 수준은 샘플에서 유의하게 높았다. 1highres.jpg &quot;/&gt; 그림 1 :. BSAS 방법의 도식이 방법에서는 게놈 DNA는 유라에 unmethylation의 사이토를 수정할 변환 중아입니다. 그 후, PCR 동안 이러한 유라는 thymines로 변경됩니다. PCR 증폭은 관심 영역에 대해 유도 및 높은 단지 이러한 시퀀스 풍부하게된다. 얻어진 PCR 증폭 산물은 간단한 공정을 통해 tagmentation 듀얼 인덱싱 라이브러리로 이루어진다. 그 후 라이브러리는 벤치 탑 차세대 시퀀서에 순서가되고, 순서는 시토신의는베이스 고유의 방식으로 결정된다 실리에서 변환 된 기준 서열과 퍼센트 메틸화에 매핑되는 읽습니다. <img alt="그림 2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> 그림 2 :. 관심 지역의 실리 중아 변환의 모든 게놈 기준에서 관심의 모든 영역이 SELEC 될 수 있습니다실리코 중아 변환에 대한 테드. 비의 CpG 사이토는 티민의 기준 순서로 대체됩니다. 의 CpG 사이토는 중아에서 사이토가 참조를 변환 남아있다. <img alt="그림 3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> 그림 3 :. 프라이머 배치 바이 설 파이트 PCR 프라이머가 실리 중아에 대해 설계는 레퍼런스 시퀀스로 변환. 뇌관은 CG 디 뉴클레오티드를 중첩하지 않도록 설계되거나 CG 디 뉴클레오티드에 인접 설계되어야한다. <img alt="그림 4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> 그림 4 : 높은과 낮은 품질의 PCR 증폭 산물과 염기 서열 라이브러리의 예 대표 electropherogram 추적 및 젤 쇼 (A) 하나 고와 transposome 매개 라이브러리 생성을위한 최적의 증폭.농도 제품입니다.이 증폭 물에서 생성 (B) 라이브러리 (C) 품질이 낮은 PCR 증폭 산물의 크기, 농도가 낮을 수있다. 고농도 심지어 크기 분포를 보여 주며, 여러 제품 및 / 또는 프라이머 이량 체를 포함한다. 이러한 낮은 품질의 증폭 산물은 (D)으로 이어질 낮은 농도와 낮은 크기 분포와 라이브러리 생성을 실패합니다. <img alt="그림 5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> 그림 5 : 메틸화 컨트롤에서 시퀀싱 출력의 예. 인 실리코 (A)가 된 CpG 사이트와 참조 순서가 빨간색으로 강조 변환. 매핑 0 % 메틸화 컨트롤의 CpG 사이트에서 선택한 영역 표시 thymines (탐욕 강조 표시) 매핑을 읽습니다. 매핑 (B) 100 % 메틸화 컨트롤의 CpG 사이트에서 시토신의 (파란색 강조 표시) 매핑을 보여 읽습니다. 도 6 :. 메틸화 컨트롤 범위에서 메틸화 정량 0 %에서 100 %까지 메틸화 비율 (N = 3 / 비)로 혼합 전체 게놈 메틸화 BSAS 컨트롤을 사용하여 정량 하였다. 정량화 대 예상의 정량을 플로팅 메틸화 제어 정량의 선형성을 보여줍니다. 모든 컨트롤에 걸쳐, 메틸화 정량의 높은 정밀도가 있었다. <img alt="그림 7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> 도 7 : 조직 샘플로부터의 페어링 된 DNA 메틸화와 유전자 발현 분석의 예. (A) 마우스 소뇌와 망막 조직에서 로돕신 상대 mRNA 발현. (B) 소뇌 및 망막 DNA (*** p &lt;0.001, 파라 메트릭 t-test를) 사이에 BSAS에 의해 정량화 로돕신 평균 프로모터 메틸화. &lt;마우스 소뇌와 망막 DNA의 전사 시작 사이트 [TSS])를 기준으로 로돕신 프로모터 (+6 -244에 걸쳐 강한&gt; C)의 CpG 사이트 별 메틸화 수준.

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.

DNA 메틸화 분석 차세대 시퀀싱 대상

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 조회수.  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.

비디오: 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.

에 각인 유전자의 DNA Methylation의 결정 Arabidopsis 배젖

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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생물학

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.

의 DNA Methylation : Bisulphite의 변경 및 분석

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.

형광 - 현미경 검사와 차세대 장면 : Organelle 무결성에 관련된 유전자의 식별을위한 유용한 도구

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.

16S 리보솜 RNA 유전자 증폭 산물의 차세대 시퀀싱

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.

자료 쌍 해상도에서 DNA 메틸화의 평가에 대한 표현 바이 설 파이트 시퀀싱을 감소 향상

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ : 아데닌 - 메틸화 된 DNA 단편의 높은 처리량 시퀀싱에 의해 단백질 DNA 상호 작용의 게놈 넓은 매핑

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

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.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

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

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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.

위하여-Seq: Domainome 라이브러리 건설, 유효성 검사 및 페이지 표시 및 다음 세대 시퀀싱 선택 프로토콜

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