Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing

Ethical considerations

The human cell lines, laboratory animals and microbial stocks used in this study do not require ethics approval in accordance with Okinawa Institute of Science & Technology Graduate University (OIST)’s Animal Care and Use Committee and Japan Society for the Promotion of Science (JSPS) guidelines. All procedures regarding the welfare and handling of organisms meet ARRIVE guidelines for the care and use of experimental animals. In particular, Drosophila were anaesthetized with carbon dioxide (CO₂) prior to mechanical cell disruption.

Sample preparation

Samples were manually homogenized either by grinding using a simple tube-based mortar and pestle (single whole eclosed fruit fly) or freeze-thawing five times (cultured cyanobacteria and algae). The human cell culture was not subjected to mechanical disruption.

Three newly eclosed single X7 Drosophila melanogaster (RRID:BDSC_5478) were isolated in vials containing food but no yeast for 18 hours, put to sleep using CO₂, and then individually ground gently in 200 μl ice-cold extraction buffer (50mM Tris-Cl pH 8.0, 100 mM NaCl, 2.5 mM EDTA, 0.5% SDS, 0.2 mg/ml Proteinase-K, 1% 2-mercaptoethanol) with a tube-based mortar & pestle on ice. The homogenized extract was incubated overnight for 18 hours at 55°C. The cell lysate was centrifuged at 11,000g (Tomy, MX-302) for 10 minutes at 4°C and 200 μl of the cleared lysate used for robotic extraction.

Cultured human embryonic kidney cells, HEK293 (RRID: CVCL_45), were dissociated from a confluent tissue culture flask with 0.25% Trypsin-EDTA for 3 minutes at 37°C (Gibco, 25200056), counted on a hemocytometer, centrifuged at 800g for 5 minutes and washed with 10 ml phosphate buffered saline (PBS). 1 × 10⁶ cells were drawn off and spun again at 800g for 5 minutes. The PBS was aspirated, and the cell pellet was resuspended in extraction buffer 50 mM Tris-Cl pH 8.0, 100 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5% SDS, 0.2 mg/ml Proteinase-K, 1% 2-mercaptoethanol and incubated for 2 hours at 55°C. The cell lysate was processed immediately by centrifugation at 11,000g for 10 minutes at 4°C and 200 μl of the cleared lysate used for robotic extraction.

The cyanobacterium Synechoccous sp. was centrifuged at 2,500g for 5 minutes from a one-month-old culture; the resulting pellet was washed with filtered autoclaved seawater three times and gently resuspended in 250 μl of extraction buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS, 2% 2-mercaptoethanol) and frozen overnight at –80°C. The next morning the cyanobacteria were thawed on ice, an equal volume of 5% SDS, 50 mM EDTA & 50 mM Tris-Cl pH 8 was mixed by inversion and subjected to four more freeze/thaw cycles. Mixed algal cultures of 6 × 10⁶ Chaetoceros calcitrans, 1 × 10⁷ Isochrysis sp. and 1 × 10⁶ Rhinomonas reticulata were centrifuged similarly from a one-month-old culture; the resulting pellet was washed with filtered autoclaved seawater three times and gently resuspended in 250 μl of ice-cold extraction buffer (100 mM Tris-Cl pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2% cetyltrimethylammonium bromide, 0.1% polyvinylpolypyrrolidone, 2% 2-mercaptoethanol) and then frozen overnight at –80°C. The next morning, the algal extractions were thawed on ice and subjected to four more freeze/thaw cycles. All extractions were centrifuged at 4°C for 10 min at 15,000g and 200 μl of the cleared lysate used for robotic extraction.

Robot-assisted organic extraction

A workflow based on a modified organic solvent based back-extraction method (Giles et al., 1980) was programmed into the LabDroid with Robotic Biology Institute's BioPortal Protocol Maker graphical user interface (Liu and Plessy, 2022b). Though the robot is capable of processing 14 samples in a run, the development was carried out with three samples. The procedural schematic and robotic workflow are outlined in Figure 2A and B, respectively. To start, 200 μl of cleared lysate was transferred to a new 2 ml tube (Eppendorf 0030 120.094) and used for the robot-assisted protocol. 200 μl of Phenol, chloroform and isoamyl alcohol (25:24:1, Sigma-Aldrich P3803) was used for the first two robot “hand-mixed” organic extractions. The closed-capped tubes were mixed by the robot’s arm/manipulators by inversion at a rate of one every 3 seconds, for a total of 5 minutes. These were later centrifuged at 15,000g for 1 minute at 4°C. The first extraction aqueous phase was transferred to a new 2 ml Eppendorf tube awaiting at 4°C. Remaining DNA left behind was back-extracted by the same means after addition of fresh extraction buffer to the phenol phase and the second aqueous phase was pooled in same 2 ml tube with the first aqueous phase. 400 μl of chloroform (Wako 3034-02603) was saturated with 10 mM Tris-Cl pH 8.5 1 mM EDTA and then used to extract the pooled aqueous phases. These were “hand-mixed” by inversion with the robotic arm and centrifuged similarly as the phenol extractions. The 325 μl of the aqueous phase from the chloroform extraction was then transferred to a new 1.5 ml centrifuge tube (Eppendorf 0030 125.215). 650 μl of 100% ethanol and 33 μl of 3 M sodium acetate pH 5.2 (Sigma Aldrich, S7899) was added, “hand-mixed” for 5 minutes and centrifuged at 15,000g for 30 minutes at 4°C. 50 μg glycogen (Thermo, R0561) was added to the final 1.5 ml tubes before the start of the robotic run to help visualize pellets after precipitation and centrifugation. After centrifugation, the tubes were placed into a 4°C block awaiting sample recovery. An entire run was video-captured, and the steps are highlighted at key points described in this manuscript as a visual demonstration of the robot’s movement (below).

Figure 2. Graphical description of the LabDroid’s workflow.

A, Schematic representation of manual (blue text) and robotic (black text) manipulations. Circular black arrows indicate robotic “hand-mixing” inversions and black arrows, centrifugation. B, Automated workflow of extraction protocol. For both panels, green arrows indicate transfer to new tube and red arrow heads, disposal of waste.

Pulsed field electrophoresis and long-read sequencing

Subsequently, the ethanol supernatant from the last automated step was removed from the pelleted DNA by manual aspiration. The pellets were carefully washed with 70% ethanol, removed by manual aspiration and air dried for 5 minutes at the bench. The washed DNA pellets were gently resuspended in 15 μl 10 mM Tris-Cl and 1 mM EDTA and quantitated with Qubit high-sensitivity double-stranded DNA reagents and fluorometer (Thermo Fisher, Q32851). 10 ng of the sample was used for pulsed-field (Agilent, FP-1002-0275) or standard capillary electrophoresis Tape Station (Agilent, 5067-5365). 1000 ng of gDNA was used for adapter ligation (Oxford Nanopore Technologies, ONT LSK-109). Between 40–500 ng of adapter ligated DNA was loaded onto a MinION R9.4 flow cell (ONT FLO-MIN106D) and allowed to run for 18-24 hours. N50 metrics were determined with the MinKNOW software (ONT).

Length of genomic DNA from automated preparations

Mammalian, algal, cyanobacteria and drosophila genomic DNAs were prepared using the organic extraction protocol programmed for Maholo. Long-read sequence data were confirmed after analysis of completed Nanopore MinION using the standard sequencing N50 metric for the length of average read: N50 values were 30-40 kb for algal samples, 20 kb for cyanobacteria and 16 kb for drosophila (Table 1). Representative results from the reverse-field gel electrophoresis genomic analysis kits are shown for Synechecoccus and Rhinomonas preparations (Figure 3A & B, respectively). For all preparations, fragments ranged in size between 50 kb and 200 kb, similar to manually prepared DNA in the hands of an experienced technician.

Figure 3. Representative Femto Pulse fragment length results from: A, cyanobacteria (Synechococcus sp.) and B, microalgae (Rhinomonas reticulata).

Troubleshooting problems during protocol development

We solved practical problems iteratively in order to implement a standard phenol-chloroform DNA extraction method on the Maholo LabDroid. Below we describe the major obstacles we encountered and their solutions (Table 2).

Organic solvents can affect the physical properties of plastics even though the effects may be imperceptible to the naked eye. Here, we found that prolonged exposure of Eppendorf tubes to phenol and chloroform caused enough changes in their dimensions for the LabDroid’s manipulators to sense and abort the run. We overcame this issue by adding aliquoted phenol and chloroform just before the start of the organic extractions. Furthermore, we found that the manipulators would drop the tubes owing to the changes in tube elasticities after centrifuging with organic solvent. The force to grip the tubes was increased during tube removal from the centrifuge to resolve this problem.

As the LabDroid does not detect the position of the liquid interfaces, it relies on the preprogrammed volumes to be correct. Mixture of organic solvents and water can vary in final volume depending on how much water was originally present, for instance through contamination of solvent aliquots by the moisture in the air. Variations in volumes reduce the precision of the robot's pipetting capabilities relative to the phase interface. It also impaired the robot’s handling of the centrifugation step since the slight imbalance caused individual tubes to rotate about their vertical axes as they balanced the mass distribution in the rotor. This changed the position of the hinge at the end of the run which prevented the robot from removing the rotated tube from the rotor. To ensure volume reproducibility, chloroform was saturated with 10 mM Tris-Cl pH 7.5 and 1 mM EDTA (TE) the night before (1:1 phenol and chloroform was purchased pre-equilibrated with TE pH 8) to prevent any aqueous phase volume loss due to the solvent’s slight water solubility. Finally, we also programmed the LabDroid to change the pipette tips after each aliquot of solvent was added, as the actual volume of solvent transferred by the tips was slightly greater after the tip had been pre-wetted.

The greatest hurdle was the collection of aqueous phases after extraction and centrifugal phase separation; this occurs three times in the protocol. The LabDroid initially was fitted with a brand of Bluetooth actuated liquid handling micro-pipettors that could not aspirate the aqueous phase without disturbing the organic/aqueous interface. As a result, the aqueous phases were contaminated with solvent which caused purity problems downstream. This was solved when new electronic pipettors (Viaflow, Integra Biosciences), capable of slower rates of aspiration, were outfitted to replace the earlier model. The aspiration offset height (the distance between the end of the pipette’s tip to the bottom of the tube) was also increased by 1 mm to avoid disturbing the organic/aqueous interface. As this increased the dead volume, we added a back-extraction step to collect the leftover aqueous phase.

Attempts at extending the protocol beyond organic extraction steps

Currently, the automated protocol ends with the pelleted DNA in salted ethanol solution, which is suitable for long-term DNA storage. We tried to incorporate a last step to precipitate the pellet in 70% ethanol and resuspend it in TE. We were unsuccessful as the pellet dislodged after the wash centrifugation. While a skilled technician can carefully remove supernatant in the presence of a floating pellet, a robot with no visual sensors cannot. To overcome this, we tried partial aspiration of the 70% ethanol wash to prevent loss of the pellet and drying 50-70 μl of remaning supernatant with a vacuum centrifuge for 40 minutes at room temperature at 0.02 bar, but we found this method partially degraded preparations. As a result, we decided to leave out the 70% ethanol wash from the automated protocol.

Underlying data

Zenodo: Nanopore MinION Run Metrics and genomic DNA fragment size analysis data from automated phenol-chloroform extractions (RBI LabDroid Maholo). https://doi.org/10.5281/zenodo.5921205 (Liu and Plessy, 2022a).

This project contains the following files:

Zenodo: Job files for automated phenol-chloroform extraction (RBI LabDroid Maholo). https://doi.org/10.5281/zenodo.5733820 (Liu and Plessy, 2022b).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Extended data

figshare: Video of robot-assisted organic extraction of DNA (RBI LabDroid Maholo). https://doi.org/10.6084/m9.figshare.19189466.v1 (Liu, Villar-Briones, Luscombe, and Plessy, 2022).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).