三代测序原理与数据文件简介(SMRT+Nanopore)
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 1578 收藏 2
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 Molecules
Abstract
Introduction
Methods
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
(Fig. A) A single molecule of DNA template-bound Φ29 DNA polymerase is immobilized at the bottom of a ZMW。
(Fig. B) Schematic event sequence of the phospholinked dNTP incorporation cycle, with a corresponding expected time trace of detected fluorescence intensity from the ZMW. (标记的dNTP插入的示意图,以及相对应的从ZMW检测到的荧光强度的预期时间轨迹)
(1) A phospholinked nucleotide forms a cognate association with the template in the polymerase active site, (在聚合酶的活性位点dNTP与模板互补配对)
(2) causing an elevation of the fluorescence output on the corresponding color channel. (催化反应导致相应的)
(3) Phosphodiester bond formation liberates the dye-linker-pyrophosphate product, which diffuses out of the ZMW, thus ending the fluorescence pulse. (磷酸二酯键的形成释放了染料-连接剂-焦磷酸盐产物,该产物从ZMW扩散出去,从而终止了荧光脉冲)
(4) The polymerase translocates to the next position, and (5) the next cognate nucleotide binds the active site beginning the subsequent pulse. ((4)聚合酶转移到下一个位置,(5)下一个同源核苷酸与活性位点结合,开始随后的脉冲。)
PS:A fluorescence pulse is produced by the polymerase retaining the cognate nucleotide with its colorcoded fluorophore in the detection region of the ZMW. It lasts for a period governed principally by the rate of catalysis, and ends upon cleavage of the dye-linker-pyrophosphate group, which quickly diffuses from the ZMW detection region.(荧光脉冲由聚合酶产生,该聚合酶将同源核苷酸及其彩色荧光团保留在ZMW的检测区域。它持续一段主要由催化速率控制的时间,并在染料链接基焦磷酸盐裂解时结束,该裂解迅速从ZMW检测区域扩散。)
The duration of the fluorophore retention is much longer than the time scales associated with diffusion (2 to 10 ms) or noncognate sampling (<1 ms), which manifest as a low and constant background signal. ……The sequence of fluorescence pulses recorded in the plot of intensity versus time is referred to as a read. (荧光信号存在时间长短,区分匹配碱基与游离碱基)
Using synthetic DNA to illustrate approach
To illustrate the principle of our approach to DNA sequencing, we used a synthetic, linear,
single-stranded DNA template with a two-base artificial sequence pattern. (为了说明我们的DNA测序方法的原理,我们使用了一个合成的,线性的,单链DNA模板,但是我们只使用A555-dCTP and A647-dGTP也就是G和C两种dNTP)
(Fig. A) GC分布的区域
(Fig. B&C)箭头表示引发聚合反应的催化金属离子的加入。
potential of long-read DNA sequencing
we performed a similar two-base signature sequence pattern experiment using a single-stranded 72-base circular DNA template (Fig. 3A). The template was designed such that cytosines were present on only half of the circle, and guanines on the other half. Φ29 DNA polymerase is highly processive (>70,000 bases) without cofactors in bulk reactions. It will carry out multiple laps of DNA strand-displacement synthesis around the circular template. (我们使用单链72碱基圆环状DNA模板进行了双碱基序列模式实验(图3A)。模板的设计使得胞核嘧啶只出现在一半的圆环上,鸟嘌呤则出现在另一半的圆环上。Φ29 DNA聚合酶在没有辅助因子的情况下反应具有高持续性(> 70000个碱基)。它将围绕圆形模板进行多次strand-displacement类型的DNA合成。)
Occasional pauses in DNA polymerization activity are visible as gaps in the trace. The total synthesized DNA length as a function of time (Fig. 3C) shows periods of different persistent polymerization rates during these long reads. Two characteristic polymerization rates of ~2 bases/s and ~4 bases/s were determined, suggesting the existence of different long-lived polymerase modes that occasionally and suddenly interconvert. No spatial correlation in the polymerase speed was observed across a ZMW array. Pulse characteristics underlying these two states were statistically identical, with the exception of a decreased interpulse duration for the faster state (fig. S2). Similar behavior was also observed using different combinations of the fluorophores and bases and for templates with different sequences (fig. S3), which implies that these states are specific neither to the phospholinked dNTPs used nor to the sequence context. (可以看到一些合成中的停顿。总合成DNA长度作为时间的函数(图3C)显示了这些长读过程中不同的持续聚合速率。测定了2个碱基/s和4个碱基/s的两种典型聚合速率,表明存在着不同的持续合成模式,它们之间偶尔会突然发生相互转化。聚合酶速度在ZMW阵列上没有空间相关性。这两种状态下的脉冲特征在统计学上是相同的,除了更快状态下的脉冲间隔时间缩短(图S2)。使用不同的荧光团和碱基组合以及不同序列的模板也观察到类似的行为(图S3),这意味着这些状态既不是特定于所使用的受磷dNTPs,也不是特定于序列上下文)
14个循环,1008个碱基,72个一个周期。一个周期上12C个12G。注意只是每个碱基脉冲时间波动很小,而不是每种dNTP的脉冲时间相同)
hence, this sequencing approach maintains accuracy irrespective of read length.
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。
想法:SMRT测序的矛盾点,如果形成Continuous Long Reads (CLR),那么随机误差严重,如果short reads形成Circular Consensus Sequencing (CCS),准确度可以保障,但是又失去了long reads的优势。
官方SMRT流程文档
SMRT下机文件示例
官方软件
Oxford Nanopore单纳米孔测序
- Nanopore DNA sequencing offers the possibility of a label-free, single-molecule approach that can be performed without the need for sample amplification. (纳米孔DNA测序提供了一种无标签、单分子方法的可能性,无需样品扩增即可进行。)
- Like second-generation systems, nanopore technology is amenable to parallelization, and several cost estimates place nanopore sequencing in the $1,000 range for a complete human genome. (一个完整的人类基因组进行纳米孔测序需要花费1000美元。/第二代技术将单倍体人类基因组重新测序到高质量的全部成本(包括仪器、样品制备和人工)目前在10万至100万美元左右。)
- Furthermore, because the sequence quality should be constant throughout a read, long reads from single molecules of DNA will be possible by using nanopores, offering many advantages including the possibility of de novo sequencing, the high-resolution analysis of chromosomal structure variation, and long-range haplotype mapping. (使用纳米孔可以长时间读取单个DNA分子,这提供了许多优势,包括从头测序的可能性、染色体结构变异的高分辨率分析和远程单倍型映射)
Nanopore sequencing principle
Structure
Pipeline
Determine the bases & Errors
Reference
Data
PS
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