Summary

Automated Gel Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

Published: April 17, 2015
doi:

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

1. Raccolta Acqua e Filtrazione Raccogliere campioni di acqua dolce provenienti da vari siti (Figura 1). Passare campioni attraverso una serie di filtri: 1 micron filtro, filtro 0,2 micron, e 30 kDa taglio del filtro a flusso tangenziale sistematicamente eucariotica separata, particelle di dimensioni batteriche e virali, rispettivamente. Nota: Solo la purificazione e analisi del materiale recuperato sui 0,2 micron filtri (batteri a vita libera) è descritta in questa relazione, ma appr…

Representative Results

Concentrazione DNA, e valutazione della qualità Concentrazioni DNA batterico variava ,01-0,11 ng ​​/ ml per campione dei vari siti di bacino (Tabella 1). DNA isolato da frazione batterica aveva A 260/280 e 260/230 A rapporti di 1,4-1,8 e 0,3-1,6 rispettivamente. Mentre alcuni dei coefficienti A 260/230 erano relativamente bassa, nessuna inibizione apparente è stata osservata nella biblioteca preparato e altre applicazio…

Discussion

La presenza di dimeri adattatori e raggruppamento di piccole dimensioni inserto preferenzialmente essere sequenziati in piattaforme attuali rappresentano una diminuzione delle rese utilizzabili e sotto-utilizzo della capacità di 15 dell'apparecchiatura. L'uso di metodi branello battitori combinati con un approccio basato trasposone-per preparare librerie può comportare più di taglio DNA rispetto ad altri metodi di estrazione 4,5. Tuttavia, tutti i metodi di estrazione e biblioteca prepar…

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

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Cite This Article
Uyaguari-Diaz, M. I., Slobodan, J. R., Nesbitt, M. J., Croxen, M. A., Isaac-Renton, J., Prystajecky, N. A., Tang, P. Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples. J. Vis. Exp. (98), e52685, doi:10.3791/52685 (2015).

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