Overview | Culturing | DNA extraction | DNA quantification | Library preparation | Data analysis | Laboratory Manual | Costs

Introduction

The concentration, molecular weight (MW) and integrity of the extracted DNA is important for WGS. There are a number of methods to do this, but Qubit and Agilent TapeStation is recommended. The use of Nanodrop or other spectrophotometric methods for estimating nucleic acid concentrations is not recommended, as they can be unreliable.

DNA input requirements

Concentration

The ONT Rapid Barcoding 96 kit (SQK-RBK110.96) used in this pipeline requires ~50-400 ng input DNA. More or less can impact the efficiency of the sequencing and is likely to result in lower yield. It is also important, when multiplexing (e.g. using multiple barcoded samples), that the input quantity for each sample is as even as possible, to avoid uneven reading across the samples. The Rapid Barcoding Kit requires an input of 9 µl for each sample, which means obtaining 5-44 ng/µl is necessary.

Molecular weight

ONT sequencing utilises long read technology, and sequencing is much more efficient with longer strands of nucleic acid. Therefore, the ONT Rapid Barcoding kit (SQK-RBK110.96) used in this pipeline requires an average MW of at least 30 kb for each sample. Often DNA extraction may yield samples with a MW of 50,000 kb or more, and when multiplexing, it is important to ensure the molecular weights are all similar to ensure even sequencing across all samples. If there are large differences in MW between samples (e.g. one is 30 kb and one is 50 kb), normalising by calculating the fmol for each sample can help to ensure even sequencing. 

Integrity

don’t want contamination with salts/proteins/RNA. Minimal degradation. gDNA should have a high MW band (>12kb) with little/no evidence of degradation (smearing). Degraded genomic DNA may result in over-fragmentation of DNA to below the desired size range and poor quality libraries. A good library should be free of any smaller or larger products than the target size for the library preparation technique. 

Estimation of DNA concentration

The quantity, quality and MW of the extracted DNA must be quantified, using the methods detailed below to ensure it is sufficient for WGS analysis. The recommended method for quantity estimation is the Qubit, and the TapeStation or gel electrophoresis should be used to identify the quality and MW of the DNA (whilst the Tapestation also identifies quantity, the Qubit is considered more accurate and overloading or underloading DNA onto the ONT flow cell can result in suboptimal sequencing).

Qubit (or other flourescence based kits) - quantification

Nanodrop - quantification (not very accurate) and quality

Tapestation (or other automated gel electrophoresis machines) - quantification, size/MW and quality

Bioanalyser -  quantification, size/MW and quality

Gel electrophoresis - size/MW and quality

Qubit

The Qubit dsDNA Assay Kits are designed for accurate DNA quantification and are highly selective for double-stranded DNA (dsDNA) over RNA.

Equipment/Reagents

-          Qubit® dsDNA Assay HS Kit. This is the high sensitivity (HS) kit with quantitation range of 0.2-100ng. Note: The broad range (BR) kit (quantitation range of 2-1000ng) can also be used if required.

  • Qubit® dsDNA HS Reagent (Component A)
  • Qubit® dsDNA HS Buffer (Component B)
  • Qubit® dsDNA HS Standard #1 (Component C)
  • Qubit® dsDNA HS Standard #2 (Component D)

- Disposable plastic container for mixing the Qubit® working solution

- Thin-wall, clear, 0.5ml PCR tubes. Acceptable tubes include Qubit® assay tubes (Life Technologies Cat No. Q32856) or Axygen® PCR-05-C tubes (VWR Cat No. 10011-830).

- Qubit Fluorometer

Storage of reagents: The Qubit® dsDNA HS Reagent and Buffer must be stored at room temperature (22-28°C) and the Qubit® DNA standards must be stored at 4°C. The Qubit Reagent must be protected from light. When stored as directed kits are stable for 6 months.

Process

  1. Set up the required number of 0.5 ml tubes for the samples and standards (2 tubes) and label the lids with the sample name.
  2. Prepare Qubit® working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in the Qubit® dsDNA HS Buffer in a clean plastic tube. The final volume in each tube is 200 μL, so prepare enough working solution for all the samples and the two standards. For example, for 8 samples and 2 standards make 2 ml of working solution by diluting 1 μL of reagent into 1990 μL of buffer.
  3. Add 190 μL of Qubit® working solution to each of the standard tubes, and then add 10 µL of each Qubit® standard to the appropriate tube and mix by vortex for 2-3 seconds. Be careful not to create bubbles.
  4. Add Qubit® working solution to individual sample tubes so that the final volume including the sample is 200 μL. The volume of the sample can be between 1-20 μL, so the volume of working solution can be adjusted accordingly e.g. 1:199 or 2:198 or 10:190
  5. Add each sample to the appropriate assay tubes and mix by vortex for 2-3 seconds.
  6. Allow all tubes to incubate at room temperature for 2 minutes – the samples are now ready to be read on the fluorometer.
  7. On the home screen of the Qubit® Fluorometer, press DNA and then select dsDNA High Sensitivity as the assay type. The standards screen is displayed.
  8. Press Yes to read the standards. Insert the tube containing Standard #1 into the sample chamber, close the lid and press Read. When the reading is complete (~3 seconds) remove the standard. Insert the tube containing Standard #2 into the sample chamber, close the lid and press Read. When the reading is completed remove the standard. When the calibration is complete the instrument displays the Sample screen.
  9. Insert a sample tube into the sample chamber, close the lid and press Read. When the reading is complete (approx. 3 seconds) remove the tube. The instrument displays the results on the screen. The first value displayed is the concentration in the assay tube.
    1. To find out the concentration in the original sample, press Calculate Stock Conc. The Dilution Calculator Screen is displayed. Select the volume of the sample that you added to the assay tube, once selected the Qubit® Fluorometer calculates the original sample concentration using the volume and the measured assay concentration.
    2. Press Read Next Sample and repeat steps 9 and 10 for all remaining samples.

NOTE: if the DNA concentration obtained is above the upper limit of the test kit, dilute the sample 1:10 and repeat testing to obtain an absolute concentration value.

Tapestation

Equipment/reagents

-          Genomic DNA Ladder (store at 2 – 8°C)

-          Genomic DNA Sample Buffer (store at 2 – 8°C)

-          Genomic DNA ScreenTape (store at 2 – 8°C. If you run less than 16 lanes, store used ScreenTape device upright at 2 – 8°C for a maximum of 2 weeks. Never freeze).

-          Loading tips (5067- 5152 or 5067- 5153)

-          Optical Tube 8x Strip (401428)

-          Optical Cap 8x Strip (401425) or 96-well Sample Plates (5067- 5150) and 96-well Plate Foil Seal (5067- 5154).

-          Vortex mixer

-          Volumetric pipette

-          Microcentrifuge

Process

  1. Launch the 2200 TapeStation Controller Software.
  2. Load Genomic DNA ScreenTape device and loading tips into the 2200 TapeStation instrument.
  3. Allow reagents to equilibrate at room temperature for 30 min.
  4. Vortex mix reagents before use.
  5. In the first well in the tube (A1) Prepare ladder by mixing 10 μL Genomic DNA Sample Buffer with 1 μL Genomic DNA Ladder (Use a fresh ladder for each run. No electronic ladder is available for the Genomic DNA assay)
  6. Prepare sample(s) by mixing 10 μL Genomic DNA Sample Buffer with 1 μL genomic DNA sample (10 – 100 ng/μL) in the wells following ladder (B1, C1, D1 etc.).
  7. Spin down, then vortex using IKA vortex and adaptor at 2000 rpm for 1 min.
  8. Spin down to position the sample at the bottom of the tube.
  9. Load samples into the 2200 TapeStation instrument.
  10. Select the required samples on the 2200 TapeStation Controller Software.
  11. Click Start.

Nanodrop

Manual:  Thermo Scientific NanoDrop 2000/2000c Spectrophotometer V1.0 User Manual

NanoDrop 2000 is a spectrophotometer that can be used to quantify the DNA and protein content in a 1-2 µl sample. Nucleic acids (such as DNA) and proteins absorb light at the 260 nm and 280 nm respectively.  RNA is a common contaminant in genomic DNA extracts and nanodrop cannot distinguish between DNA and RNA very well, hence less accurate for quantification of DNA for library preparation purposes. It also can be used to measure the purity of DNA extracts through measuring the A260/A280 ratio (recommended values 1.8 – 2) and A260/A230 (recommended value (2.0 – 2.2).

Process:

  1. Launch the NanoDrop 2000 software interface.
  2. Raise the sampling arm, apply 2 μL blank solution onto the lower pedestal, carefully lower down the sampling arm and initiate blank measurement using the software on the PC.
  3. When the measurement is complete, raise the sampling arm and wipe the blank from both the upper and lower pedestals using a clean lint-free laboratory wipe.
  4. Apply 2 μL sample solution onto the lower pedestal and initiate a spectral measurement.
  5. Wipe the sample from both the upper and lower pedestals using a clean lint-free laboratory wipe before you apply the next sample.

Cleaning of the instrument

Between measurements

Upon completion of each sample measurement, wipe the sample from both the upper and lower

pedestals with a clean, dry, lint-free lab wipe, to prevent sample carryover and avoid residue build-up. It is not necessary to clean with distilled water (dH2O) between samples.

Between users

A final cleaning of both measurement surfaces with dH2O is recommended before the first sample

measurement and after the last sample measurement.

Additional cleaning

When a more rigorous cleaning protocol is required (i.e. dried proteins) substitute 0.5M HCl for the dH2O in the procedure above. Follow with 3-5 μL of dH2O.

Decontamination

A sanitizing solution, such as a 0.5% solution of sodium hypochlorite (1:10 dilution of common commercial bleach solutions – freshly prepared), may be used to ensure that no biological material is present.

Bioanalyser

Manual: Agilent Bioanalyser

  • DNA 12000 kits are designed for the sizing and quantitation of double-stranded DNA
  • fragments from 100 to 12000 bp.
  • For accurate determination of DNA concentration, the total amount of DNA in sample must be between 0.5–50 ng/μL.  If concentration is excessively high, dilute to 0.5–50 ng/μL.
  • Can analyse 12 samples/chip, chip cannot be reused.

Equipment/reagents

-          Agilent 2100 Bioanalyzer instrument

-          Chip priming station (065-4401)

-          IKA vortex mixer

-          Agilent DNA 12000 Kit (5067-1508)

-          Pipettes (10 μL, 100 μL and 1000 μL) with compatible tips

-          0.5 mL microcentrifuge tubes for sample preparation

-          Microcentrifuge

N.B. Keep all reagents and reagent mixes refrigerated at 4 °C when not in use to avoid poor results caused by reagent decomposition. Protect dye and dye mixtures from light. Remove light covers only when pipetting. Dye decomposes when exposed to light.

Process

Preparing the Gel-Dye Mix

1)      Allow DNA dye concentrate (blue) and DNA gel matrix (red) to equilibrate to room temperature for 30 min.

2)      Vortex DNA dye concentrate (blue) and add 25 μL of the dye to a DNA gel matrix vial (red).

3)      Vortex solution well and spin down. Transfer to spin filter tube.

4)      Centrifuge at 1500 x g ± 20 % for 10 min. Protect solution from light. Store at 4°C.

Loading the Gel-Dye Mix

1)         Allow the gel-dye mix to equilibrate to room temperature for 30 min before use.

2)         Clean the Bioanalyzer electrodes before using them

3)         Put a new DNA chip on the chip priming station

4)         Pipette 9μL of gel-dye mix in the well marked with a shaded G.

Note: Pipette in the bottom corner of the wells. Make sure the gel-dye mix is at RT. Wait if not.

5)         Make sure the plunger is set to 1mL and close the chip priming station.

6)         Pres the plunger until it is held by the clip.

7)         Wait for exactly 30 second and release the clip.

8)         Wait for 5 seconds and then slowly pull up the plunger back to the 1 ml position.

9)         Open the chip priming station and pipette 9μL into the other two wells marked G.

10)     Pipette 5μL of marker (green tube) in all 12 sample and ladder wells.

11)     Pipette 1μL of DNA ladder (yellow tube) in the ladder well.

12)     Load 1μL of the DNA sample in the first well and 1μL of MinION buffer in the second.

13)     In the remaining 10 sample wells, load 1μL of NFW. Do not leave any well empty.

14)     Put the chip in the Agilent Vortex and vortex for 1 minute.

15)     Load the chip into the Bioanalyzer and start the run within 5 minutes.

Gel electrophoresis

Conventional agarose gel electrophoresis employs a static field and can resolve DNA fragments up to 50 kilobases (kb)

Equipment/reagents

-          Agarose powder

-          Tris/Borate/EDTA buffer (TBE buffer)

-          GelRed® (Biotium) or Ethidium Bromide

-          High MW DNA ladder (Size range: 0.5 kb to 48.5 kb)

-          Loading/tracking dye mixture

-          Paraffin film or 0.2 mL PCR tubes

-          Casting tray/combs

-          250 ml glass flasks

-          Balance

-          Microwave

-          Electrophoresis battery

-          Power supply

-          UV lamb/camera

Process

1)      Prepare 0.5x Tris/Borate/EDTA buffer (TBE buffer) by diluting 50 mL TBE buffer (often supplied as 10x concentrate) in 950 mL distilled water.

2)      Prepare 0.7% w/v agarose solution in in appropriate volume of 0.5x TBE: 50 or 100 mL according to the capacity of the casting tray in a flask that is several times larger than the volume required. Do not cover or close the flask/bottle

3)      Boil the solution for 1-2 minutes to dissolve the agarose, swirl the solution periodically until the solution turns clear, note the flask is hot use appropriate heat insulating gloves.

4)      Cool down by swirling the flask under running tap water

5)      Once the gel has reaches about 55°C, add the appropriate volume of the DNA staining dye: Ethidium bromide (final concentration 0.3 µg/mL in the agarose gel) or the safer GelRed® (10,000x concentrate in DMSO), swirl gently. Note that ethidium bromide is a mutagenic agent so handle with care and use appropriate PPE.

6)      Pour the agarose gel/dye mixture slowly in the casting tray with the appropriate comb, avoid creating bubbles, leave the gel to cast ~30 minutes.

7)      Place the tray with the gel in the electrophoresis tank and fill it with 0.5x TBE buffer (electrophoresis buffer) to the maximum level mark.

8)      Carefully pull up the comb to create the wells in the agarose gel.

9)      Mix 5µL of each DNA sample with 1µL of sample loading dye/tracking dye mixture (6x) by pipetting up and down on a paraffin film or in PCR tubes.

10)   Load the samples into the wells, note the order of samples, reserve one well for the DNA Ladder.

11)   Load high MW DNA Ladder in the gel

12)   Cover the electrophoresis tank with the lid, make sure that the electrical leads are connected to the battery’s electrodes (power supply) correctly.

13)   Switch on the battery note the current is running through the buffer in the electrophoresis chamber by observing bubbles at the electrodes. Electrophoresis is usually done at 100V for 0.5-1 hour at room temperature.

14)   At the end of the run switch off the power supply, remove the gel tray from the electrophoresis chamber, wipe the outside to get rid of excess buffer

15)   Image the gel under UV lamp.