Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

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 degraded^1-3 and the presence of inhibitors of PCR^4-6.

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 data^7,8. 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 insert^9,10. There are numerous methods^11,12 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 DNA^13,14. 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. Water Collection and Filtration

1. Collect freshwater samples from various sites (Figure 1). Pass samples through a series of filters: 1 µm filter, 0.2 µm filter, and 30 kDa cutoff tangential flow filter to systematically separate eukaryotic, bacterial and viral sized particles, respectively.
   Note: Only the purification and analysis of the material recovered on the 0.2 µm filters (free-living bacteria) is described in this report, but similar approaches can be used for the other particles recovered from the filters.

2. Bacterial Concentration, DNA Extraction and Purification

1. Remove the 0.2 µm membrane filter(s) from the water filtration system (schematic diagram, Figure 2). Fold the 0.2 µm disc filter in half and place it into a 50 ml tube with the open side up. Add 5 ml of 1x phosphate buffered solution (PBS) and 0.01% Tween, pH 7.4 into the tube. If more than one filter is used during the process employ a different tube per filter. Store tube(s) with filter at 4 °C until processed. Otherwise, proceed to step 2.2.
2. Remove the 0.2 µm membrane filter from the 50 ml tube with sterile forceps. Place filter(s) on a petri dish (leave the PBS buffer in the tube).
   1. With sterile forceps and sterile scissors, cut the filter into small strips (1 cm x 1 cm). Disinfect forceps and scissors by soaking in 70% isopropanol, wiping with a DNA surface decontaminant and rinsing with ultrapure water of type 1 between different samples.
3. Place the filter cut into strips back into the same 50 ml tube. Add 10 ml of 1x PBS and 0.01% Tween, pH 7.4 to the filter so there is a total of 15 ml of buffer in the 50 ml tube.
4. Add 6 clean tungsten beads (3 mm diameter) to each 50 ml tube, cover the tube with Parafilm, and vortex vigorously for 20 min (use 50 ml tubes vortex adaptor). If multiple filters are processed from one sample, transfer and pool all the homogenate into a clean 50 ml tube. Centrifuge the tubes at 3,300 x g for 15 min at 4 °C.
5. Resuspend the pellet and aliquot bacterial cells (~1 ml aliquots) into 1.7 ml microcentrifuge tubes. Note that there will be 5 microcentrifuge tubes per sample.
6. Spin down the microcentrifuge tubes at 10,000 x g for 10 min, and remove most of the supernatant down to ~200 µl mark.
7. Store the samples at -80 °C, or proceed to extract bacterial DNA by using a commercially available DNA isolation kit as per the manufacturer’s instructions. During bacterial DNA extraction use either PBS or water as a negative extraction control, and E. coli as a positive extraction control.
8. Because DNA from multiple tubes are being extracted, pool parallel sub-aliquots from the same sample into one 2 ml tube. Then conduct an O/N precipitation by using a solution comprised of 0.1 volumes of 3 M sodium acetate, 2 volumes of 100% ethanol, and 5 µl of 5 µg/µl linear acrylamide. Mix well and store samples at -80 °C.
9. Centrifuge at maximum speed (17,000 x g) for 30 min at 4 °C. Wash pellet with 1 ml of ice-cold 70% ethanol, air dry in a biosafety cabinet for no more than 5 min and resuspend DNA pellet in 34 µl of 10 mM Tris-Cl, pH 8.5.
   Note: Linear acrylamide will help to visualize DNA pellet after the O/N precipitation.
10. Determine DNA quantity and quality using a high sensitivity fluorescent nucleic acid quantitation assay and a microvolume spectrophotometer, respectively, following the manufacturers’ instructions (See Table of Materials).
    Note: If multiple samples are prepared at the time, an ultrasensitive fluorescent nucleic acid assay for quantifying dsDNA and plate-based fluorometer/spectrophotometer can be used instead.

3. DNA Library Preparation

1. Normalize genomic bacterial DNA extracted from water samples to a concentration of ~0.2 ng/μl.
2. Enzymatically fragment one nanogram of bacterial DNA using a transposon-based method (tagmentation). Add adapter and index sequences onto fragmented DNA following the manufacturer’s instructions. Subject tagmented samples to 12 cycles of PCR as indicated in the manufacturer’s instructions, at which point the sequencing indexes are incorporated.

4. Automated Gel Size Selection

1. Skip standard post-PCR clean-up step described in the manufacturer’s instructions (See Table of Materials). Size select post-PCR samples directly using an automated gel-based size selection platform.
   1. Add 3.5 μl of loading buffer to each sample.
   2. Following the manufacturer's protocol, load the samples onto the electrophoresis workstation, specifying a size-selection target of 500-800 bp, in a 300 μl extraction volume, using a pre-cast 1.5% agarose cassette.
   3. Concentrate the raw output material (300 μl) down to 25 μl via ethanol precipitation.
      Note: Alternatively, use the electrophoresis workstation to automate concentration for larger numbers of samples via filter plate and vacuum manifold.
      1. Precipitate the raw elution volume using 0.1 volumes of 3 M sodium acetate, 2 volumes of 100% ethanol and 5 μl of 5 µg/µl linear acrylamide. Mix well, place samples at -80 °C O/N. Centrifuge samples at 17,000 x g for 30 min.
      2. Discard supernatants and proceed to wash DNA pellet with 1 ml of ice-cold 70% ethanol.
      3. Centrifuge samples again at 17,000 x g for 30 min, discard supernatants, air dry, and finally resuspend DNA pellet in 25 μl of 10 mM Tris-Cl, pH 8.5.
2. (Optional) Use 1 μl of DNA libraries size selected with the automated gel size selection platform to make a five-fold dilution using Tris-EDTA buffer solution, pH 8.0. Analyze libraries using a chip-based capillary electrophoresis instrument to analyze dsDNA quality according to manufacturer’s instructions.

5. Library Normalization and Sequencing

1. Proceed to normalize samples using library normalization beads (included in the transposon-based library preparation kit) as described in the manufacturer’s preparation guide.
   1. Alternatively, measure dsDNA libraries using a high sensitivity fluorescent nucleic acid quantitation assay, followed by pooling at equimolar ratios to reach a final dsDNA concentration of 2 nM. Proceed to denature libraries using 10 μl of pooled libraries and 10 μl of 0.2 N NaOH, incubate for 5 min at RT. Dilute denatured libraries using hybridization buffer (included with the transposon-based preparation kit) to a final volume of 1 ml and concentration of 11.5 pM.
2. Load samples in a next-generation sequencing platform by following the manufacturer’s standard sequencing protocols.

Results

DNA Concentration, and Quality Assessment

Bacterial DNA concentrations ranged from 0.01 to 0.11 ng/ml per water sample of the different watershed sites (Table 1). DNA isolated from bacterial fraction had A_260/280 and A_260/230 ratios of 1.4 to 1.8 and 0.3 to 1.6, respectively. While some of the A_260/230 ratios were relatively low, no apparent inhibition was observed in the prepared library and other downstream applications. Thes...

Discussion

The presence of adapter dimers and clustering of small insert sizes preferentially being sequenced in current platforms represent a decrease in useable yields and under-utilization of the equipment’s capacity¹⁵. The use of bead beating methods combined with a transposon-based approach to prepare libraries may result in more DNA shearing compared to other methods of extraction^4,5. Nevertheless, all methods of extraction and library preparation potentially introduce biases to the distribution of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.
20x 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, 120 V (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