環境水試料から調製した次世代シーケンシングライブラリの品質を向上させるために自動化されたゲルサイズの選択
Summary
This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.
Abstract
Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments.
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.
Introduction
Metagenomics involves the sequencing of all the genetic material in a sample to characterize the microbial communities present. It is a complex and expensive process which involves the conversion of extracted nucleic acids into DNA libraries followed by next-generation sequencing. High quality libraries are essential for maximal data output and accurate metagenomics analysis. Environmental samples, such as water samples, often pose significant challenges to generating high quality libraries, due to low amounts of DNA that may also be degraded1-3 and the presence of inhibitors of PCR4-6.
High quality libraries ideally consist of longer segments of DNA within a narrow range of lengths. In order to maximize the amount of useful data generated per sequencing run, the length of the DNA in the library should be at least as long as the maximum read length of the sequencing method being used. When using a sequencing-by-synthesis technology such as the Illumina MiSeq, the size of the DNA fragments affects the efficiency at which clusters are generated on the flow cell. For instance, when a library contains both shorter and longer DNA fragments, the shorter ones will be over-represented in the sequencing data7,8. In contrast, a library with similarly sized DNA fragments will be proportionally represented in the sequencing data. Many library preparation kits use ligation-based methods to add adapters to the DNA fragments and size selection is necessary to remove adapter dimers that do not contain an insert9,10. There are numerous methods11,12 to achieve this but the one technique that gives the most consistent results is the electrophoretic separation of DNA followed by the recovery of the desired lengths of DNA13,14. This process can be performed manually for a small number of samples, but when faced with processing hundreds of samples, automated solutions are required. The currently available platforms for automated gel size selection are low throughput and new platforms are needed to process large numbers of samples for sequencing. The Ranger Technology can be integrated with existing liquid handling workstations to enable the use of agarose gel electrophoresis for size selection and analytical purposes on a scale that satisfies today’s high throughput environment.
Protocol
Representative Results
Discussion
アダプターダイマーと優先的に現在のプラットフォームで配列決定されている小型のインサートサイズのクラスタリングの存在は、使用可能な収率で、かつアンダー利用設備の容量15の減少を表している。ライブラリーを調製するためのトランスポゾンベースのアプローチと組み合わさビーズビーティング法の使用は、抽出4,5の他の方法と比較して、よりDNAの剪断を生じ得る?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by Genome BC, Genome Canada, and Coastal Genomics. The authors thank Kirby Cronin and Michael Chan for their help in sample collection and processing. We also would like to acknowledge Thea Van Rossum and Dr. Fiona Brinkman for bioinformatics assistance.
Materials
| Peristaltic pump Masterflex P/S 1400 Series | Thermo Scientific | 1400-1620 | |
| 0.2 µm Supor Membrane | VWR | CA28143-969 | Pall Corporation, Ann Harbor, MI |
| Tungsten carbide beads 3 mm (200) | Qiagen | 69997 | |
| Isopropyl alcohol 70% | Jedmon Products | 825751 | Healthcare Plus |
| DNA away | VWR | 7010 | Molecular BioProducts, Inc. San Diego, CA |
| Milli-Q water purification system | Fisher Scientific | ZMQS6VF0Y | Merck Millipore. This system has been discontinued. |
| 20 X PBS pH 7.5 | VWR | E703-1L | Amresco, Inc., Solon, OH |
| Tween 20 | Fisher Scientific | BP337-100 | Fisher chemicals |
| Vortex adapter for 2 (50 ml) tubes | VWR | 13000-V1-50 | MoBio, Carlsbad, CA |
| Vortex-Genie 2, 120V (Model G560) | VWR | SI-0236 | Scientific Industries, Inc. |
| Beckman Centrifuge | Raeyco Lab Equipment Systems Management Ltd | Model J-6B | |
| PowerLyzer Powersoil DNA isolation kit | VWR | 12855-100 | MoBio, Carlsbad, CA |
| Vortex adapter for 24 (1.5-2 ml) tubes | VWR | 13000-V1-24 | MoBio, Carlsbad, CA |
| Microfuge 18 centrifuge | Beckman Coulter | 367160 | |
| Nimbus Select workstation with Ranger Technology | Hamilton Robotics | 92720-01 | Includes the liquid handling workstation and integrated Ranger Tech (electrophoresis hardware) |
| Ranger reagent kit | Coastal Genomics | CG-10600-150-12-21 | Includes loading buffer and cassettes |
| Ethyl alcohol (anhydrous) | Commercial Alcohols | P016EAAN | Greenfield Ethanol |
| Sodium acetate | Sigma-Aldrich | S2889-250G | |
| Linear acrylamide (5 mg/ml) | Life Technologies | AM9520 | Ambion |
| Eppendorf refrigerated centrifuge | Raeyco Lab Equipment Systems Management Ltd. | 5417R | |
| Buffer EB (250 ml) | Qiagen | 19086 | |
| NanoDrop 1000 Spectrophotometer | Thermo Scientific | ND-1000 | |
| Qubit fluorometer | Life Technologies | Q32857 | Invitrogen. This product has been discontinued. |
| Qubit dsDNA HS assay kit | Life Technologies | Q32854 | Invitrogen |
| High sensitivity DNA reagent | Agilent Technologies | 5067-4626 | |
| High sensitivity DNA chips | Agilent Technologies | 5067-4626 | |
| Agilent 2011 Bioanalyzer | Agilent Technologies | G2938B | |
| Nextera XT DNA sample preparation kit | Illumina | FC-131-1024 | |
| Nextera XT index kit | Illumina | FC-131-1001 | |
| MiSeq reagent kit v2 (500-cycles) | Illumina | MS-102-2003 | |
| Miseq system | Illumina | SY-410-1003 |
References
- Siuda, W., Chrost, R. J. Concentration and susceptibility of dissolved DNA for enzyme degradation in lake water – some methodological remarks. Aquatic Microbial Ecology. 21, 195-201 (2000).
- Ficetola, G. F., Miaud, C., Pompanon, F., Taberlet, P. Species detection using environmental DNA from water samples. Biology letters. 4, 423-425 (2008).
- Liles, M. R., et al. Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms. Applied and environmental microbiology. 74, 3302-3305 (2008).
- Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. Extraction of high molecular weight DNA from microbial mats. BioTechniques. 49, 631-640 (2010).
- Tebbe, C. C., Vahjen, W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Applied and environmental microbiology. 59, 2657-2665 (1993).
- Solonenko, S. A., et al. Sequencing platform and library preparation choices impact viral metagenomes. BMC genomics. 14, 320 (2013).
- Aird, D., et al. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome biology. 12, R18 (2011).
- Quail, M. A., Swerdlow, H., Turner, D. J., et al. Improved protocols for the illumina genome analyzer sequencing system. Current protocols in human genetics / editorial board, Jonathan L. Haines .. [et al.]. 18, 18 (2009).
- Head, S. R., et al. Library construction for next-generation sequencing: overviews and challenges. BioTechniques. 56, 61-64, 66, 68 (2014).
- Dijk, E. L., Jaszczyszyn, Y., Thermes, C. Library preparation methods for next-generation sequencing: tone down the bias. Experimental cell research. 322, 12-20 (2014).
- Grunenwald, H., Baas, B., Caruccio, N., Syed, F. Rapid, high-throughput library preparation for next-generation sequencing. Nature Methods. 7, (2010).
- Sambrook, J., Russell, D. W. . Molecular cloning : a laboratory manual. , (2001).
- Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments. , (2012).
- Quail, M. A., et al. A large genome center’s improvements to the Illumina sequencing system. Nature methods. 5, 1005-1010 (2008).
- Marine, R., et al. Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA. Applied and environmental microbiology. 77, 8071-8079 (2011).
- Rohland, N., Reich, D. Cost-effective high-throughput DNA sequencing libraries for multiplexed target capture. Genome research. 22, 939-946 (2012).
- Lamble, S., et al. Improved workflows for high throughput library preparation using the transposome-based nextera system. Bmc Biotechnology. 13, (2013).
- Picelli, S., et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome research. , (2014).
- Rhodes, J., Beale, M. A., Fisher, M. C. Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq. PloS One. 9, e113501 (2014).
