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Abstract
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Biology

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Published: March 22, 2016
doi:
10.3791/53840
, ,
1Department of Cancer Immunology and AIDS,Dana-Farber Cancer Institute, 2Chromatin Structure and Mobile DNA,The Francis Crick Institute

Summary

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.

Abstract

在本地和全球尺度上表现出逆转录病毒整合签名的偏好。这里，我们提出了（1）的生成用连接介导的PCR（LM-PCR）扩增和新一代测序（NGS）逆转录病毒整合位点的不同的库的详细的协议;（2）绘制的每个病毒 – 的基因组位置使用BEDTools交界处，和（3）分析统计相关数据主机。从感染的细胞中提取的基因组DNA是通过消化用限制性酶或通过超声处理分段。合适的DNA末端修复后，双链连接子连接到DNA末端，和半巢式PCR是使用这两个病毒的长末端重复（LTR）端部和连接接头DNA互补的引物进行的。 PCR引物携带NGS期间用于DNA聚类所需的序列，否定为单独的适配器连接的要求。质量控制（QC）下进行，以评估DNA片段大小分布和适应呃之前NGS DNA结合。序列输出文件过滤含有LTR-读取，并确定LTR和连接器的序列裁剪掉。修剪宿主细胞序列被映射到使用BLAT参照基因组和被过滤为最小97％的同一性的参照基因组中的独特点。独特的整合位点是审查相邻核苷酸（nt）的顺序和分配相对于不同的基因特性。使用该协议，高复杂度的整合位点文库可从基因组DNA在三天构成。该包括易感组织培养细胞至整合位点分析的外源病毒感染的整个协议可以因此在大约一到两周进行。这项技术的应用近期涉及到艾滋病毒感染患者的整合位点的纵向分析。

Introduction

病毒DNA（VDNA）到宿主细胞基因组中的整合是在逆转录病毒的生命周期的一个重要步骤。积分是由病毒酶整合酶（IN），其执行，导致建立稳定地插入原病毒1的两个不同的催化过程来完成的。在亚基接合是通过逆转录生成的线性VDNA的端部，形成具有VDNA高阶intasome端通过一个IN多聚体2-4保持在一起。在切割中称作3'-处理的处理顺序的3'从不变5'-CA-3下游VDNA的端部“，留下凹入3'在每个VDNA末端5-8活性羟基结束。该intasome随后导入细胞核作为大型组件的主机的一部分，并称为preintegration复合物（PIC），9-11的病毒蛋白。遇到细胞靶DNA（T-DNA）后，使用VDNA 3'-羟基GRO起坐裂解TDNA顶部和底部股线以交错的方式，同时通过链转移12,13的过程中加入VDNA到TDNA 5'磷酸基团。

在本地和全球尺度上表现出逆转录病毒整合位点的偏好。在当地，共识的整合位点包括了从VDNA插入位点14,15大约五到十个基点的上游和下游跨越弱保守的回文T-DNA序列。全球范围内，逆转录病毒针对具体的染色质注释16。有七种不同的逆转录病毒属 – 通过ε，伦蒂和spuma阿尔法。慢病毒，包括HIV-1，有利于积极转录基因17机构内部整合，而gammaretroviruses优先整合到转录起始位点（起始位置跟）和有源增强地区18-20。与之形成鲜明对比，泡沫病毒强烈向heterochrom偏见ATIC区域，如基因贫乏椎板相关领域21。当地TDNA基地偏好由IN和T-DNA 13,22,23之间核蛋白的接触特定网络决定的很大一部分。对于慢病毒和gammaretroviruses，相对于基因组注释集成是通过中和同源细胞因子24-27之间的相互作用支配很大一部分。改变IN-T-DNA相互作用网络13,22,23,28的细节，扰乱或重新工程宿主因子交互25-27,29-32是行之有效的战略分别重新定位在本地和全球层面的整合。

用来编目逆转录病毒整合位点的DNA测序方法的功率在过去几十年的极大增加。整合位点用费力净化和手工克隆技术产生只是根据研究33,34独特的网站屈指可数回收创举。LTR宿主DNA结的LM-PCR扩增与映射单个整合位点，以转化领域人类和小鼠草案基因组，与来自外源组织培养细胞的感染中回收网的增加至几百到上千17数量的能力的结合18。 LM-PCR检测方法NGS的更近的组合已派出图书馆深度跃然而出。具体而言，焦磷酸测序，得到的独特的集成网站30,35-38数以万计的量级，而库测序通过使用DNA聚类可以产生数以百万计的独特序列19-21,39的。在这里，我们描述了扩增和测序使用DNA集群NGS逆转录病毒整合位点的优化LM-PCR方法。该方法结合了所需的适配器序列引入PCR引物，因此直接进入扩增的DNA分子，从而排除了要求用于向sequen之前一个额外的适配器连接步骤庆安40。在生物信息学分析管道中，从LTR宿主DNA结到的独特的整合位点的映射到相关的基因组特征的原始测序数据的解析，也一般地描述。按照这一领域36,38,41-43从以前的方法确定的协议的优先级，自定义脚本可以开发，以帮助在生物信息学管道的具体步骤的完成。该实用程序和协议的灵敏度通过扩增，测序和映射从在感染的1.0（MOI），以及一个滴定系列该DNA的近似多重感染的组织培养细胞中的HIV-1的整合位点与代表性数据示出通过未感染的细胞的DNA中稀释5倍的步骤为1的最大稀释度：15625，得到的6.4×10 -5的近似等效惯性矩。

Protocol

1.生成病毒原液注意：此协议的湿式清洗台方面的流程图在图1中描绘的病毒原料生产和组织培养细胞的随后感染的细节通常将适用于不同类型的逆转录病毒。对于一些实验，靶细胞可能不表达内源性病毒受体（多个），并且在这种情况下，假型逆转录病毒颗粒携带异源病毒包膜糖蛋白的结构， 例如 ，从水泡性口炎病毒（VSV-G）对G糖蛋白，会需要感染44,4…

Representative Results

表4列出一个代表性实验的结果，以示出用于从感染细胞的培养物中回收整合位点的NGS的灵敏度。未感染的细胞的DNA被用于串联从其中在平均每个细胞包含一个积分40的感染稀释的基因组DNA。 15625：在五个步骤的1的最大稀释制备稀释液。在滴定系列的基因组DNA，然后通过超声处理片段化或通过用限制性内切核酸酶消化MseI和BglⅡ，随后的LM-PCR的独特整合…

Discussion

一种用于逆转录病毒整合位点的分析协议，从通过基因组分布模式映射的初始病毒感染步骤，进行说明。这个协议是适用于任何反转录病毒和任何可感染的细胞类型。此外，该测定管道是相当敏感，以回收的从基因组DNA等效的系列稀释独特的整合位点令人满意编号以与6.4×10 -5的MOI启动的感染的可能性。当从可能含有低病毒载量，其中只有细胞的一小部分会窝藏集成原病​​毒感染的患者?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们非常感谢我们的同事斯蒂芬·休斯和亨利·莱文的意见，这是建立逆转录病毒整合位点的测序NGS协议在恩格尔曼实验的关键。这项工作是由美国国立卫生研究院授予AI039394和​​AI052014（以ANE）和AI060354（哈佛大学艾滋病研究中心）的支持。

Materials

DMEM Gibco 11965-084 Standard cell culture medium, compatible with HEK293T cells
Fetal Bovine Serum Thermo Scientific SH 30088.03 Different lots of serum may need to be pre-screened for optimal viral production
Penicillin/Streptomycin Corning 30-002-Cl Antibiotics to be added to DMEM
Phosphate-buffered saline Mediatech 21-040-CV Used to wash cells
Trypsin EDTA Corning 25-053-CI Used to detach adherent cells from tissue culture plates
PolyJet SignaGen Laboratories SL100688 DNA transfection reagent
0.45 µm Filters Thermo Scientific 09-740-35B Used to filter virus particle-containing cell culture media
Turbo DNase Ambion AM2239 Used to degrade carryover plasmid DNA from virus stocks
HIV-1 p24 Antigen Capture Assay ABL Inc. 5447 Used to quantify yield of virus production
DNeasy Blood & Tissue Kit Qiagen 69506 Used to purify genomic DNA from cells
Sonicator Covaris S2 With this model of sonicator perform two rounds of duty cycle, 5%; intensity, 3; cycles per burst, 200; time, 80 sec
Nuclease-Free Water GeneMate G-3250-125 Commercially-available water is recommended to reduce the possibility of sample cross-contamination
QIAQuick PCR Purification Kit Qiagen 28106 Used to purify DNA during library construction
End-It DNA End-Repair Kit Epicentre ER81050 Used to repair DNA ends of sonicated DNA samples
Klenow Fragment (3'-5' exo–) New England Biolabs (NEB) M0212S Used with dATP to A-tail repaired DNA fragments
dATP Thermo Scientific R0141 Deoxyadenosine triphosphate
MseI NEB R0525L Restriction endonuclease for genomic DNA cleavage
BglII NEB R0144L Restriction endonuclease to suppress amplification of upstream HIV-1 U5 sequence
T4 DNA Ligase NEB M0202L/6218 Enzyme for covalent joining of compatible DNA ends
DNA Oligonucleotides Integrated DNA Technologies custom Have the company purify the oligos. HPLC purification suffices for DNAs <30 nucleotides; PAGE purify longer DNAs 
Advantage 2 Polymerase Mix Clontech 639202 Commercial mix containing DNA polymerase for PCR
dNTPs (100 mM solutions) Thermo Scientific R0181 Dilute the four chemicals on ice with sterile water to reach the intermediate worrking concentrations of 2.5 mM each dNTP
NanoDrop Thermo Scientific NanoDrop 2000 Spectrophotometer for determination of DNA concentration
Qubit Fluorimeter Life Technologies Qubit® 3.0 Fluorometer used to confirm integration site library DNA concentration
2200 TapeStation System Agilent G2964AA Tape-based assay to confirm integration site library DNA size distribution
MiSeq Illumina SY-410-1003 Used for NGS
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Tags

Keywords: AmplificationNext-generation SequencingGenomic DNA MappingRetroviral Integration SitesPCRDNA SequencingHEK293T CellsTransfectionVirus ConcentrationP24 AntigenCell Infection
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Serrao, E., Cherepanov, P., Engelman, A. N. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites. J. Vis. Exp. (109), e53840, doi:10.3791/53840 (2016).
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