Research School of Biology, The Australian National University, Acton, ACT, Australia.
Malaghan Institute of Medical Research, Wellington, New Zealand.
Mol Ecol Resour. 2019 Jan;19(1):77-89. doi: 10.1111/1755-0998.12938. Epub 2018 Oct 5.
Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant on extracting high-quality, high-molecular-weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time-consuming and costly. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long-read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long-read sequencing in their own laboratories.
长读测序技术正在改变我们组装高度复杂基因组的能力。要充分发挥其潜力，关键是要从感兴趣的生物体内提取高质量、高分子量（HMW）的 DNA。对于便携式 MinION 测序仪来说尤其如此，由于其入门成本低，空间占用小，因此所有实验室都可以开展自己的基因组测序项目。MinION 的一个挑战是，每个小组都必须独立建立使用该仪器的有效方案，这可能既耗时又昂贵。在这里，我们提出了一个工作流程和方案，该方案基于优化从具有挑战性的植物组织中提取 DNA 作为案例研究，使我们能够在自己的实验室中建立 MinION 测序。按照所说明的工作流程，我们能够可靠且重复地获得 >6.5 Gb 的长读测序数据，平均读长为 13 kb，N50 为 26 kb。我们的方案是开源的，无需特殊设备即可在任何实验室中进行。我们还说明了一些更复杂的工作流程，如果需要，可以提高平均和平均读长。我们设想，我们建立 MinION 测序的工作流程，包括可能出现的问题的说明以及如何将其适应其他组织类型的建议，将对计划在自己的实验室中建立长读测序的其他人有用。
