PacBio vs. Oxford Nanopore sequencing

PacBio vs. Oxford Nanopore sequencing

PacBio与牛津纳米孔测序

发表于 2017年6月16日通过Bhagyashree Birla

由太平洋生物科学公司和牛津纳米孔公司开发的长读测序技术克服了研究人员短读所面临的许多限制。读可改善从头组装，转录组分析（基因同工型鉴定），并在宏基因组学领域发挥重要作用。当组装包括大片段重复区域的基因组时，较长的读段也很有用。

当前，有两个长读测序平台。为了帮助研究人员选择哪种平台在其应用中具有更大的实用性，我们比较了PacBio和Oxford Nanopore提供的整体仪器规格以及下一代测序领域中已发布的应用。

一个牛津纳米孔指责为用户提供了一个奴才/ PromethIon仪器，耗材的启动包，某些数据服务和基于社区的支持接入费

*数据不足

尽管与短读Illumina或离子测序相比，PacBio和Oxford纳米孔均产生更长的读取，但是PacBio和Oxford纳米孔测序仪的较高错误率仍然是需要解决的问题。PacBio会多次读取一个分子以生成高质量的共有数据，而Oxford Nanopore只能对一个分子进行两次测序。结果，与牛津纳米孔相比，PacBio产生的错误率更低。PacBio在诸如发现转录组复杂性和灵敏鉴定同工型等应用方面的总体性能稍好一些。另一方面，MinION可以提供更高的通量，因为纳米孔可以同时对多个分子进行测序。因此，它最适合需要大量数据的应用程序9

由于长的阅读可以提供大型的支架，从头组装是PacBio测序5的主要应用之一。尽管PacBio数据的错误率高于短读Illumina或离子测序的错误率，但增加覆盖率或混合测序可以大大提高基因组装配的准确性。PacBio测序已成功用于完成Autoethanogenum 梭状芽胞杆菌  DSM 10061（第III类）的100个重叠群的基因组图  ，这是就重复含量和重复类型而言最复杂的基因组分类。它的GC含量为31.1％，并包含重复序列，原噬菌体和9个rRNA基因操纵子。使用单个PacBio库并用两个SMRT细胞对其进行测序，即可从头组装整个基因组  与一个重叠群。当短读Illumina或离子测序单独用于同一基因组时，需要> 22个重叠群，并且每个装配体至少包含四个折叠的重复区域，PacBio装配体没有10个。

与使用Illumina测序仪12进行组装相比，PacBio测序还用于组装委陵菜11，酿酒酵母，拟南芥和黑果果蝇的叶绿体基因组。

与Sanger测序相比，PCR产品的PacBio测序可通过缩小缺口并通过发夹结构和高GC含量区域进行测序来提高当前草图基因组的质量13。

太平洋生物科学公司已开发出一种用于转录本测序的方案Iso-Seq。这包括库构建，大小选择，排序数据收集和数据处理。Iso-Seq允许直接测序高达10 kb的转录本，而无需使用参考基因​​组。Iso-Seq已用于表征参与血细胞成分14形成的可变剪接事件。这对于解释导致遗传性疾病和血液癌症的突变的影响至关重要，并且可以应用于设计策略以促进移植和再生医学。

PacBio测序的另一个主要应用是表观遗传学研究。最近的研究表明，通过PacBio测序15直接检测单个分子中的修饰，可以促进对先前无法检测到的基因组DNA修饰（如m 6 A和m 4 C）中细胞间异质性的研究。

与PacBio相比，Oxford Nanopore MinION体积小（USB拇指驱动器大小），价格适中，利用简单的文库制备且可现场携带16。这在病毒爆发等情况下很有用，在这种情况下可以使用MinIONS设置移动诊断实验室。在诸如巴西和非洲部分偏远地区，这些地方存在与运送样品进行测序相关的后勤问题，MinION可以向科研人员提供即时和实时数据。MinION最著名的临床用途是在西非病毒爆发期间现场对埃博拉病毒样本进行分析17,18。

MinION测序仪的低成本测序和便携性也使其成为教学的有用工具。它已被用于向学生提供动手实践，最近在哥伦比亚大学和加利福尼亚大学圣克鲁斯分校，每个学生都在其中进行了自己的MinION测序19。

MinION最雄心勃勃的应用可能是它有潜力检测和识别载人航天飞行中的细菌和病毒。在概念验证实验中，Castro-Wallace等人。展示了λ噬菌体基因组，大肠杆菌基因组和小鼠线粒体基因组的成功测序和从头组装。他们观察到，在国际空间站上生成的序列数据的质量以及在地球22上并行执行的控制实验中，都没有显着差异。

最近，Oxford Nanopore开发了台式仪器PromethION，该仪器可提供高通量测序，并且在设计上是模块化的。它包含48个流通池，可以单独运行或并行运行。PromethION流通池每个包含3000个通道，并产生多达40 Gb的数据。

Posted on June 16, 2017 by Bhagyashree Birla

Long-read sequencing developed by Pacific Biosciences and Oxford Nanopore overcome many of the limitations researchers face with short reads. Long reads improve de novo assembly, transcriptome analysis (gene isoform identification) and play an important role in the field of metagenomics. Longer reads are also useful when assembling genomes that include large stretches of repetitive regions.

Currently, there are two long read sequencing platforms. To help a researcher choose between which platform has greater utility for their application, we compare overall instrument specifications offered by PacBio and Oxford Nanopore, and published applications in the next-generation sequencing space.

a Oxford Nanopore charges an access fee that gives users one MinION/PromethIon instrument, a starter pack of consumables, certain data services, and community-based support

* Insufficient data

Although both PacBio and Oxford Nanopore generate longer reads compared to short read Illumina or Ion sequencing, the higher error rate of both the PacBio and Oxford Nanopore sequencers remain an issue needs addressing. Whereas PacBio reads a molecule multiple times to generate high-quality consensus data, Oxford Nanopore can only sequence a molecule twice. As a result, PacBio generates data with lower error rates compared to Oxford Nanopore. PacBio has a slightly better overall performance for applications such as the discovery of transcriptome complexity and sensitive identification of isoforms. On the other hand, MinION provides higher throughput as nanopores can sequence multiple molecules simultaneously. Hence, it is best suited for applications that require a larger amount of data9

As long reads can provide large scaffolds, de novo assembly is one of the main applications of PacBio sequencing5. Though the error rate of PacBio data is higher than that of short read Illumina or Ion sequencing, increased coverage or hybrid sequencing can greatly improve the accuracy of genome assembly. PacBio sequencing has been successfully used to finish the 100-contig draft genome of Clostridium autoethanogenum DSM 10061, a Class III, the most complex genome classification in terms of repeat content and repeat type. It has a 31.1% GC content and contains repeats, prophage, and nine copies of rRNA gene operons. Using a single PacBio library and sequencing it with two SMRT cells, an entire genome can be assembled de novo with a single contig. When short read Illumina or Ion sequencing was used alone with the same genome, >22 contigs were needed, and each of the assemblies contained at least four collapsed repeat regions, PacBio assemblies had none10.

PacBio sequencing has also been used to assemble the chloroplast genome of Potentilla micrantha11, Saccharomyces cerevisiae, Aradopsis thaliana and Drosophila melanogaster using fewer contigs and CPU time for assembly compared to assemblies using Illumina sequencers12.

PacBio sequencing of PCR products can be used to improve the quality of current draft genomes by closing gaps and sequencing through hairpin structures and areas of high GC content more efficiently than Sanger sequencing13.

Pacific Biosciences has developed a protocol, Iso-Seq, for transcript sequencing. This includes library construction, size selection, sequencing data collection, and data processing. Iso-Seq allows direct sequencing of transcripts up to 10 kb without the use of a reference genome. Iso-Seq has been used to characterize alternative splicing events involved in the formation of blood cellular components14. This is essential for interpreting the effects of mutations leading to inherited disorders and blood cancers, and can be applied to design strategies to advance transplantation and regenerative medicine.

Another major application of PacBio sequencing is in epigenetics research. Recent studies demonstrate that investigation of intercellular heterogeneity in previously undetectable genome DNA modifications (such as m6A and m4C) is facilitated by the direct detection of modifications in single molecules by PacBio sequencing15.

Compared to PacBio, the Oxford Nanopore MinION is small (size of a USB thumb drive), affordable, utilizes a simple library prep and is field portable16. This is useful in situations such as a virus outbreak where a mobile diagnostic laboratory can be set up using MinIONS. In remote regions such as parts of Brazil and Africa where there are logistical issues associated with shipping samples for sequencing, MinION can provide immediate and real-time data to scientific investigators. The most notable clinical use of MinION has been the analysis of Ebola samples on-site during the viral outbreak in West Africa17,18.

The low cost of sequencing and portability of the MinION sequencer also make it a useful tool for teaching. It has been used to provide hands-on experience to students, most recently at Columbia University and the University of California Santa Cruz, where every student performed their own MinION sequencing19.

Perhaps the most ambitious MinION application is its potential to detect and identify bacteria and viruses on manned space flights. In a proof-of-concept experiment, Castro-Wallace et al. demonstrated successful sequencing and de novo assembly of a lambda phage genome, an E. coli genome, and a mouse mitochondrial genome. They observed that there was no significant difference in the quality of sequence data generated on the International Space Station and in control experiments that were performed in parallel on Earth22.

Recently, Oxford Nanopore developed a bench-top instrument, PromethION, that provides high-throughput sequencing and is modular in design. It contains 48 flow cells that can be run individually or in parallel. The PromethION flow cells contain 3000 channels each, and produce up to 40 Gb of data.

 

References:

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