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三代测序原理与数据文件简介(SMRT+Nanopore)
一生雾梦 2019-12-03 20:48:42 分类专栏: 前沿文献分析 文章标签: 三代测序(SMS) SMRT Nanopore 生物信息 测序原理 版权 Pacific Biosciences单分子实时测序(SMRT) 原始文献——Real-Time DNA Sequencing from Single Polymerase Molecules①We detected the temporal order (时间顺序) of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays (零模波导纳米结构阵列), which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions (成千上万的单分子测序反应能够并行、同步检测). ②Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. ( 将荧光团与dNTPs末端磷酸基结合,可以连续观察上千个碱基的DNA合成,而不受空间位阻。这里可能指的是荧光基团的空间位阻不影响DNA的合成) ③Consensus sequences were generated from the single-molecule reads at 15-fold coverage (15倍的测序深度), showing a median accuracy of 99.3%(准确率中位数是99.3%), with no systematic error beyond fluorophore-dependent error rates (除了与荧光基团相关的错误率外,没有系统误差). Introduction①Sanger method: This method relies on the low error rate of DNA polymerases (发挥了DNA聚合酶自身的低错误率), but exploits neither their potential for high catalytic rates (高催化效率) nor high processivity (高持续合成能力). 如何理解:大肠杆菌的DNA 聚合酶I三个功能区,5’→3’ DNA聚合酶活性外,还有5’→3’(去除引物)和3’→5’(检查)的外切核酸酶活性。 Klenow fragment,去除5’→3’外切核酸酶活性,在二代测序合成中应用,效率更高。 Technology ▲when a fluorophore is linked to the terminal phosphate moiety (phospholinked), phosphodiester bond formation catalyzed by the DNA polymerase results in release of the fluorophore from the incorporated nucleotide, thus generating natural, unmodified DNA. (当一个荧光团与末端磷酸基连接时,DNA聚合酶催化的磷酸二酯键形成会使荧光团从合并的核苷酸中释放出来,从而生成天然的、未经修饰的DNA) ▲Φ29 DNA polymerase was selected for these studies because it is a stable, single-subunit enzyme with high speed, accuracy, and processivity (稳定的单亚基酶,具有快速、准确和持续合成能力) that efficiently uses phospholinked dNTPs. It is capable of strand-displacement DNA synthesis and has been used in whole-genome amplification, showing minimal sequencing context bias. (链置换复制模式,就是上次提到的一直循环复制的模式,也被广泛用于全基因组扩增) ▲we reported a surface chemistry that enables selective immobilization of DNA polymerase molecules in the detection zone of ZMW nanostructures with high yield. (使DNA聚合酶分子在ZMW纳米结构检测区域的选择性固定化成为可能) ▲可以测到甲基化,甲基化的碱基脉冲的时常和光谱的特征都会变化,所以可以捕捉。 Structure & Pipeline
About errors ⚠At optimal loading, the distribution is 36.8% empty ZMWs, 36.8% with just one polymerase, and 26.4% with two or more. (在最佳加载条件下,满足泊松分布,空ZMWs的分布为36.8%,只有一个聚合酶的分布为36.8%,两个或两个以上的分布为26.4%。) ⚠dark nucleotides need not be invoked as a source of error. (暗核苷酸不是错误来源,dNTP足够纯,而且脉冲时间显示应该没有碱基插入事件) ⚠In these data, errors are dominated by deletions, which stem from incorporation events or intervals between them that are too short to be reliably detected. (在这些数据中,错误主要是由删除造成的,删除源于插入事件,它们之间的间隔太短而无法可靠地检测到)图示为A555-dATP脉冲时间与被检测到的概率的比值(黑线),与A555-dATP脉冲时间(蓝线)复合后,得到右图。(从这些数据中,缺失率估计为7.8%,与所观察到的7.4%的核苷酸缺失率一致。) ⚠The majority of insertion errors were caused by dissociation of a cognate nucleotide from the active site before phosphodiester bond formation can occur, resulting in the erroneous duplication of a pulse. (大多数插入错误是由于在磷酸二酯键形成之前,同源核苷酸从活性位点解离,导致错误的重复脉冲) ⚠We have shown that with just 15 molecules, a consensus sequence with 99.3% median accuracy can be formed with no detectable sequence context bias and a uniform error profile within reads. (我们已经证明,只要有15个分子,就可以在不存在可检测序列上下文偏差的情况下形成具有99.3%中位精度的一致序列,并在reads中形成一致的错误表谱)根据上面的实验,不受上下文序列的影响,没有偏好性,只存在随机误差,所以可以通过多次实验避免。 DATA sample:DNA N6-adenine methylation in Arabidopsis thaliana
To enable sequencing of both strands, a library is constructed from double-stranded DNA (dsDNA) with a protocol similar to that used for short-read, second-generation platforms. The library preparation chemistries (SQK MAP005 and SQK MAP005.1) used in this study, contain two different adapters that are ligated to the DNA (Figure 1A). The first, the leader adapter , consists of two oligos with partial complementarity that form a Y-shaped structure once annealed. The second, the hairpin adapter , is a single oligo with internal complementarity to form a hairpin structure. Both adapters in the sequencing kit used for this study are preloaded with motor proteins that mediate the movement of DNA through the pore. Another function of the adapters is to guide the DNA fragments to the vicinity of pores via binding to tethering oligos with affinity for the polymer membrane (Figure 1B). Sequencing begins at the single-stranded 5 end of the leader adapter (Figure 1C). (为了实现对两条链的测序,用双链DNA (dsDNA)构建了一个文库。这里用到两个不同的DNA的接头(图1A)。第一个是leader adapter,由两个部分互补的oligos组成,退火后形成y形结构。第二种是hairpin adapter,是一个内部互补的单一寡聚体,形成发卡结构。本研究中使用的测序试剂盒中的两个接头都预先装载了motor蛋白,motor介导DNA通过nanopore。接头的另一个功能链接tether蛋白,引导DNA片段到达孔隙附近(图1B)。测序从leader接头的5’端开始(图1C)) Once the complementary (double-stranded) region of the leader adapter is reached, the motor protein loaded onto the leader adapter unzips the dsDNA, allowing the first strand of the DNA fragment, the template , to be passed into the nanopore one base at a time, while the sensor measures changes in the ionic current. After reaching the hairpin adapter, an additional protein, the hairpin protein , allows the complementary strand of DNA to pass through the nanopore in a similar fashion. (当互补(双链)区域的leader接头到达后,motor蛋白结合到leader接头除,并解旋双链DNA,允许的第一链DNA片段(也就是模板)的碱基依次通过纳米孔,而传感器测量离子电流的变化。到达发夹接头后,另一种蛋白质,即发夹蛋白质,允许互补的DNA链以类似的方式穿过纳米孔)这是一种2D的文库,实际上后来ONT感觉上逐渐淘汰了这种方案。实际上还有1D和1D2两种,区别就是接头不同,1D2可以让第二链紧接着通过,但是存在一定的概率。 The raw current measurements or the corresponding events, plotted over time, are referred to as a squiggle plot . The base-caller in use at this time modelled the characteristics of 45 (= 1,024) possible 5-mers and base-calling consisted of finding the optimal path (Figure 1G) through a Hidden Markov Model (HMM) of successive 5-mers using a Viterbi algorithm. (原始的电流测量或相应的事件,随着时间的推移,被称为波线图。这个时候使用的base-caller模仿的特点,通过连续隐马尔科夫模型(HMM)训练数据后来推算碱基的排列)对于1D文库准确度85%,1D2可以达到90%. [1] Rank, D., Baybayan, P., Bettman, B., Bibillo, A., Bjornson, K., Chaudhuri, B., … Turner, S. (2009). Real-Time DNA Sequencing from Single Polymerase Molecules. Science, (January), 133–138. [2] Pacbio官方文档:Pacific Biosciences Glossary of Terms [3] Pacbio官方文档:Introduction to SMRTbell™ Template Preparation [4] Pacbio官方文档:Perspective Understanding Accuracy SMRT Sequencing [5] Pacbio官方文档:Template Preparation [6] https://zhuanlan.zhihu.com/p/77547922 [7] https://en.wikipedia.org/wiki/FASTQ_format [8] Pacbio官方文档:SMRT® Analysis Barcoding Overview [9] Magi, A., Semeraro, R., Mingrino, A., Giusti, B., & D’Aurizio, R. (2017). Nanopore sequencing data analysis: State of the art, applications and challenges. Briefings in Bioinformatics, 19(6), 1256–1272. [10] Clarke, J., Wu, H. C., Jayasinghe, L., Patel, A., Reid, S., & Bayley, H. (2009). Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotechnology, 4(4), 265–270. [11] Ip CLC, Loose M, Tyson JR et al. MinION Analysis and Reference Consortium: Phase 1 data release and analysis[version 1; referees: 2 approved] F1000Research 2015, 4:1075 [12] Ip, C. L. C., Loose, M., Tyson, J. R., de Cesare, M., Brown, B. L., Jain, M., … Olsen, H. E. (2015). MinION Analysis and Reference Consortium: Phase 1 data release and analysis. F1000Research, 4. [13] https://zhuanlan.zhihu.com/p/91629114 Data[1] http://datasets.pacb.com/2013/Human10x/READS/index.html [2] https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM2157793 PSPacbio Inc (2004年建立,基于康奈尔的研究,以半导体和光子技术结合生物科技,Illumina在18年11月1日收购了Pacbio) & Oxford Nanopore Ltd (牛津大学化学院的Hagan Bayley教授等人建立于2005年) |
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