全基因组对真核生物转录误差的监测
Summary
该协议为研究人员提供了一种新的工具来监测多模型生物体中转录的真实性。
Abstract
基因信息的忠实表达需要准确的转录。令人惊讶的是, 对控制转录的真实性的机制知之甚少。为了填补这一科学知识的空白, 我们最近优化了循环测序检测, 以检测整个转录的酿酒酵母、果蝇和秀丽的转录错误. 线虫。该协议将为研究人员提供一个强大的新工具, 用于映射真核细胞中转录错误的景观, 以便能够在前所未有的细节上阐明控制转录的真实性的机制。
Introduction
基因组提供了精确的生物生命蓝图。为了正确地实施这一蓝图, 对基因组进行精确的转录是很重要的。然而, 转录不太可能是错误的自由。例如, 长期以来已知的 RNA 聚合酶在体外1、2中很容易出错, 最近发现它们在体内犯下错误, 以及3、5、6,特别是面对 DNA 损伤7,8,9,10。这些观察结果表明, 在所有活细胞中, 转录错误不断发生, 这表明它们可能是突变蛋白的有力来源。
这一过程, 称为转录诱变, 不同于经典诱变的两种方式。首先, 与基因突变相比, 转录错误影响有丝分裂和有丝分裂后细胞, 因为它们不依赖 DNA 复制。因此, 研究影响转录保真度的机制, 将对有丝分裂和有丝分裂后细胞的突变负荷提供有价值的见解。有趣的是, 转录错误最近被牵涉到促进蛋白质聚集11,12,13 , 并已被推测为两种致癌作用10和细菌耐药性的发展14。
第二, 与基因突变相比, 转录错误是暂时性的。它们的暂时存在特别具有挑战性, 因为它使转录错误极其难以检测。例如, 虽然几个实验室已经为研究转录诱变设计了有价值的记者化验, 但这些化验只能够测量在有限数量的上下文和模型有机体4,15的转录误差。为了克服这些局限性, 许多研究人员已经转向 rna 测序技术 (rna 序列), 理论上允许在任何物种的转录中记录转录错误。然而, 这些研究容易混淆的图书馆建设工件, 如反向转录错误, PCR 放大错误, 和容易出错的性质排序本身。例如, 反向 transcriptases 每隔2万个基元就会提交大约一个错误, 而 RNA 聚合酶 (RNAPs) 则每30万个基5、6将只进行一个错误。由于反向转录的错误率只会使 rna 聚合酶在细胞内的错误率相形见绌, 所以在传统的 rna 序列数据中, 很难区分真实的转录错误和由图书馆准备产生的伪影。图 1a)。
为了解决这个问题, 我们开发了一个优化版本的圆排序 (Cirseq, 或 C 序列从今以后) 化验5,16。这种检测允许用户检测转录错误和 RNA 中的其他罕见变种在整个转录5。循环测序法具有这个名称, 因为这个检测的关键步骤围绕 RNA circularization。一旦 RNA 目标被发送, 他们是反向转录在一个滚动圈的方式, 以产生线性 cDNA 分子, 包含许多副本相同的 RNA 模板。如果其中一个模板中存在错误, 则该错误也会出现在 cDNA 分子中包含的每一个重复项中。相反, 由反向转录、PCR 放大或测序引入的错误倾向于随机产生, 因此只会出现一次或两次重复。因此, 通过为每个 cDNA 分子生成一个一致的序列, 并区分随机错误和所有重复发生的错误, 库构造工件可以有效地与真实的转录错误分离 (图 1b)。
如果使用得当, C 序列分析可以用来准确地检测 RNA 的基替换, 插入和删除的速率, 在整个转录的任何物种 (例如, 见遍历和 Ochman17)。例如, 我们使用了 C 序列分析法, 提供了全基因组的测量在酵母、果蝇和秀丽线虫中转录的错误率, 单碱分辨率为5。(未发表的意见)。最初用于精确序列化 RNA 病毒的人群, 这种优化版本的 C 序列分析已经简化, 以尽量减少苛刻的条件, 在图书馆的准备工作, 有助于图书馆建设工件。此外, 通过使用一些商业上可用的套件, 该检测的吞吐量大大提高, 以及其用户友好性。如果使用得当, 这种检测可以准确地检测到数以千计的转录错误每复制, 从而大大提高了以前的研究6。总的来说, 这种方法提供了一个强大的工具, 研究转录突变, 并将允许用户获得新的洞察力的机制, 控制转录的真实性在广泛的有机体。
Protocol
Representative Results
Discussion
在这里, 我们描述了一个优化的协议, 以准备 C 序列库, 以检测转录错误的酿酒酵母,果蝇, 和秀丽线虫。该协议比现有的协议和替代技术具有许多优点。
在过去的15年中, 许多记者系统已经开发, 依靠荧光素酶7,8或创可氧合酶3,4,15,<sup cla…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这份出版物是由赠款 T32ES019851 (Fritsch)、R01AG054641 和一位遥远的青年调查员赠款 (m. Vermulst) 提供的资金所实现的。
Materials
| RiboPure RNA purification kit | ThermoFisher | AM1926 | Total RNA purification |
| Genelute mRNA purification kit | Sigma-Aldrich | MRN70-1KT | mRNA purification |
| Nuclease-free Water | Ambion | AM9937 | Elution and dilution |
| Ambion RNase III | ThermoFisher | AM2290 | RNA fragmentation |
| T4 RNA Ligase 1 (ssRNA Ligase) | New England Biolabs | M0204S | RNA circularization |
| Ribolock | ThermoFisher | EO0381 | RNase inhibitor |
| SuperScript III Reverse Transcriptase | ThermoFisher | 18080044 | Rolling circle reverse transcription |
| 10 mM dNTP mix | ThermoFisher | 18427013 | Rolling circle reverse transcription |
| Random hexamers (50 ng/µl) | ThermoFisher | N8080127 | Rolling circle reverse transcription |
| NEB Second Strand Synthesis Module | New England Biolabs | E6111S | Second Strand Synthesis |
| NEBNext Ultra DNA Library Prep Kit for Illumina | New England Biolabs | E7370S | cDNA library preparation |
| NEB Next index primers | NEB | E7335S | Multiplex PCR primers |
| Oligo Clean & Concentrator | Zymo Research | D4061 | Clean up of RNA and DNA samples |
| DynaMag-2 Magnet | ThermoFisher | 12321D | Magnetic bead purification |
| AMPure XP beads | Beckman Coulter | A63881 | Magnetic bead purification |
| Eppendorf 5424 Microcentrifuge | FisherScientific | 05-403-93 | centrifugation |
| INCU-Shaker 10L | Benchmark Scientific | H1010 | Cell culture |
| T100 Thermal Cycler | BIO RAD | 1861096 | Medium to High temperature cycling conditions |
| PTC-200 Thermal Cycler | GMI | 8252-30-0001 | Low temperature cycling conditions |
| RNase Away | Molecular Bioproducts | 700S-11 | Sterilization |
| 50 ml Centrifuge Tube | Corning | 430290 | Nuclease-free |
| 15 ml Centrifuge Tube | Corning | 430052 | Nuclease-free |
| Eppendorf tubes | USA Scientific | 1615-5500 | Nuclease-free |
| 4200 Tapestation System | Agilent | G2991AA | Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples |
| High Sensitivity RNA Screen Tape | Agilent | 5067-5579 | Quality control of RNA and single stranded DNA samples |
| RNA ScreenTape Sample Buffer | Agilent | 5067-5577 | Quality control of RNA and single stranded DNA samples |
| RNA ScreenTape Ladder | Agilent | 5067-5578 | Quality control of RNA and single stranded DNA samples |
| 2100 Bioanalyzer Instrument | Agilent | G2939BA | Double stranded DNA quality control |
| High Sensitivity DNA Kit | Agilent | 5067-4626 | Quality control for double stranded cDNA samples |
| Water Bath | VWR | 462-0244 | Incubation |
| NanoDrop 2000/2000C Spectrophotometer | ThermoFisher | ND-2000C | Determination of RNA concentration |
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