下一代测序现已在整个生命科学领域得到广泛使用，但是常用的短读测序方法经常会受到GC碱基对的影响。结合短读数固有的作图模糊性，通常会导致片段化的基因组组装，从而产生了对提供更长读数，简化分析并产生更完整序列的技术的需求。利用其专有技术，Pacific Biosciences能够提供更长的读取时间，更均匀的覆盖范围和更高的准确性，支持先进的基因组学，全长转录本测序和表观遗传学。
Pacific Biosciences（PacBio）是一家位于加利福尼亚门洛帕克的测序技术提供商。该公司利用其专有的单分子实时（SMRT®）测序技术，开发了两种长读测序系统– PacBio RS II和Sequel®系统–提供了基因组，转录组和表观基因组的全面视图。PacBio产品管理高级总监Marty Badgett解释说：“我们的仪器直接在装有数千个零模波导（ZMW）的SMRT细胞中对DNA进行测序，您可以将其视为非常小的反应容器。我们将聚合酶与ZMW底部的预结合DNA模板锚定，并引入荧光标记的核苷酸。ZMW从下面照亮，衰减后的光进入每个ZMW的下部20-30 nm，当聚合酶将每个碱基整合到天然链中时，激发荧光团。我们的仪器基本上通过“监听” DNA合成过程来进行信号处理。”
“我们技术的主要优点是阅读量长，覆盖范围均匀，共识准确性高。这种准确性是由于低的测序上下文偏差，极低的系统误差和读取误差的随机性所致。例如，覆盖范围为10到20倍，我们可以实现99.9％的准确读数，通过额外的测序覆盖范围，可以很容易地将其读数提高到99.999％。我们的系统还可以容纳大约200 bp至> 10,000 bp的各种模板大小，使其适用于多种应用，包括全基因组和靶向测序。因此，事实证明，SMRT测序技术已广泛用于植物和动物科学研究，尤其是在学术实验室以及人类生物医学领域，例如HLA分型。
Cavro ADP是续集系统的核心
“我们的原始系统– PacBio RS II –每个SMRT单元具有150,000 ZMW，相当于大约55,000次读取，并且每次运行最多可处理16个单元。这听起来可能非常庞大，但是吞吐量仍然是一个限制，所以我们在2015年10月启动了Sequel系统。借助该平台，每个SMRT单元包含一百万ZMW，吞吐量提高了七倍，我们目前正在开发一个包含800万个ZMW的更高密度的SMRT电池。”
“这个OEM解决方案提供了性能，准确性，成本和高级功能的结合……这对我们的应用非常有利。”
现在，NGS协议的自动化对于许多生命科学应用而言至关重要
PacBio的一名机械设计工程师Paz Yogev补充说：“当我们开发Sequel系统时，我们希望合并PacBio RS II中使用的移液器无法提供的许多其他液体处理功能。在最终决定采用Tecan的Cavro®空气置换移液器（ADP）之前，我们研究了许多现成的解决方案，以及开发基于这些选项之一的定制移液系统的可能性。该OEM解决方案将性能，准确性，成本和高级功能（包括自动尖端检测和弹出以及液位检测）结合在一起，非常适合我们的应用。我们最初购买了几个样本单元，并建立了一个测试平台来进行比较。这既简单又便宜，使移液系统的开发非常具有成本效益。此外，我们选择使用Tecan的一次性吸头，因为从同一供应商处购买移液器和消耗品可最大程度地使一切协同工作而没有任何问题。”
“我们与Cavro ADP合作已经有好几年了，它对我们来说真的很好-它非常可靠。我们的现场服务工程师会响应客户遇到的任何问题，我们很少需要与Tecan团队联系以获得额外的支持，但是我们知道，如果需要的话，就可以在那里。” Paz总结道。
Next generation sequencing is now in widespread use throughout the life sciences sector, but the commonly used short-read sequencing methods are often subject to GC base pair bias. Combined with the inherent mapping ambiguity of the short reads, this often results in fragmented genome assemblies, creating a demand for technologies offering longer reads that simplify analysis and yield more complete sequences. Using its proprietary technology, Pacific Biosciences is able to offer longer reads, more uniform coverage and high accuracy, supporting advanced genomics, full-length transcript sequencing and epigenetics.
Pacific Biosciences (PacBio) is a sequencing technology provider based in Menlo Park, California. Using its proprietary Single Molecule, Real-Time (SMRT®) Sequencing technology, the company has developed two long-read sequencing systems – the PacBio RS II and the Sequel® System – offering a comprehensive view of genomes, transcriptomes and epigenomes. Marty Badgett, Senior Director of Product Management at PacBio, explained: “Our instruments sequence DNA directly in SMRT Cells packed with thousands of zero-mode waveguides (ZMWs), which you can think of as very small reaction vessels. We anchor the polymerase with the pre-bound DNA template at the bottom of the ZMW, and introduce fluorescently-tagged nucleotides. The ZMW is illuminated from below, and the attenuated light enters the lower 20-30 nm of each ZMW, exciting the fluorophore as the polymerase incorporates each base into the native strand. Our instrument basically carries out signal processing by ‘eavesdropping’ on the process of DNA synthesis as it happens.”
“The major advantages of our technology are the long reads, uniform coverage and high consensus accuracy. This accuracy is due to low sequencing-context bias, very low systemic error and the random nature of read errors. For instance, with 10- to 20-fold coverage we can achieve 99.9 % accurate reads, which can be readily increased to 99.999 % accuracy with additional sequencing coverage. Our system can also accommodate a broad range of template sizes – from ~200 to >10,000 bp – making it suitable for a wide variety of applications, including whole genome and targeted sequencing. As a result, SMRT Sequencing technology is proving popular for plant and animal sciences research – particularly in academic labs – as well as in the human biomedical space, for applications such as HLA typing.”
The Cavro ADP is at the heart of the Sequel System
“Our original system – the PacBio RS II – had 150,000 ZMWs per SMRT Cell, equating to about 55,000 reads, and could process up to 16 cells per run. This may sound like a very large number, but throughput was still a limitation, so we launched the Sequel System in October 2015. With this platform, each SMRT Cell contains one million ZMWs, offering a sevenfold improvement in throughput, and we’re currently working on a higher density SMRT Cell containing eight million ZMWs.”
"This OEM solution offers a combination of performance, accuracy, cost and advanced features… that is very favorable for our application."
Automation of NGS protocols is now crucial for many life sciences applications
Paz Yogev, a Staff Mechanical Design Engineer at PacBio, added: “When we were developing the Sequel System, we wanted to incorporate a number of additional liquid handling features that the pipettor used in the PacBio RS II simply couldn’t offer. We researched a number of off-the-shelf solutions – as well as the possibility of developing a customized pipetting system based on one of these options – before eventually deciding on Tecan’s Cavro® Air Displacement Pipettor (ADP). This OEM solution offers a combination of performance, accuracy, cost and advanced features – including automatic tip detection and ejection, plus liquid level detection – that is very favorable for our application. We initially bought a couple of sample units and set up a test rig to run comparisons. This was both easy and inexpensive, making development of the pipetting system very cost effective. In addition, we chose to use Tecan’s disposable tips, as buying the pipettor and consumables from the same supplier maximizes the likelihood of everything working in synergy without any issues.”
“We have been working with the Cavro ADP for several years now, and it has been really good to us – it’s very reliable. Our field service engineers respond to any problems encountered by customers, and we very rarely have to reach out to the Tecan team for extra support, but we know it’s there if we need it,” Paz concluded.
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