WORKFLOW FOR THE NUCLEIC ACID BASED IDENTIFICATION OF INSECTS USING WHOLE GENOME AMPLIFICATION AND NANOPORE SEQUENCING - KAWA
Jürg E Frey, Beatrice Frey, Daniel Frei, Morgan Gueuning, Simon Blaser, Andreas Bühlmann
Abstract
BACKGROUND
World-wide trade with plant material has dramatically increased over the past decades, and with it has the risk for accidental introduction of potential plant pests and diseases. Rapid and accurate nucleic acid based identification of such quarantine organisms has become an important tool to minimize their dispersal.
Nucleic acid based identification exploits genetic diversity. A basic tenet holds that species generally do not interbreed and hence, the level of genetic differentiation within species is generally lower than between species. For insects, this pattern of genetic diversity is being used by DNA barcoding with great success. An approximately 600 base pairs long fragment in the first half of the mitochondrially encoded cytochrome c oxidase I gene (COI) is used as a reference sequence to identify insects at the species level. So far, reference sequences for ca. 231’000 different insect species are deposited on the publicly available “Barcoding for Life Database” BOLD (http://www.barcodinglife.org/; by June 2021). Another important database especially targeting phytosanitary purposes with DNA barcodes of vouchered reference specimen is the EPPO Q-Bank (European Plant Protection Organization, https://qbank.eppo.int/).
The methodology of DNA barcoding generally relies on PCR amplification of the diagnostic COI gene fragment using a pair of primers for which the exact DNA sequence must be known. However, this information is not always available, for example in the case of so far undescribed species or in cases, where genetic variation within species affected primer sites. Furthermore, although the COI marker sequence shows an impressive degree of among species differentiation, this is not true for all species and hence, a number of important pest species cannot be differentiated based on this marker alone.
An ideal method for species identification should therefore obtain information of the best discriminating genetic region or of several genetic regions. Here, we describe a state-of-the-art method to achieve this task.
PURPOSE
The purpose of this workflow is to provide a generic method for genetic identification of potential insect quarantine species and of other especially dangerous pest species in support of the Swiss Federal Plant Protection Service. The method is marker independent and may be used with reference databases of any genetic fragment. It is based on whole genome amplification, followed by single strand nanopore sequencing and DNA barcoding based identification.
Before start
Attachments
Steps
1 Sample preparation
All samples should be stored frozen at -20°C or stored in 70% (few days at Room temperature or at 4°C in the refrigerator) until processed. Samples can be stored frozen indefinitely. Use sterile dissection equipment where appropriate. As we are mostly using crude DNA extracts with or without further cleanup for the WGA step there is a need to equilibrate tissue size with the amount of proteinase K buffer solution. The following table lists empirically established conditions:
| A | B |
|---|---|
| Homogenization Buffer (μl) | Organism (or parts thereof) |
| 50 | Thrips larva 1st and 2nd instar; small Whitefly larvae (1st or 2nd instar) or eggs; very small tissue sample |
| 100 | Thrips adult or 3rd instar; large Whitefly larvae (3rd instar) or pupae; tissue samples of corresponding size; antenna or tarsus (or part of leg) of adult tephridit fruit fly; 0.5x0.5x0.5mm sample of insect larvae |
| 200 | Drosophila adult; 1x1x1mm sample of larger insect larvae or of caterpillars |
2 DNA Extraction
Materials:
The extraction is carried out with the proteinase K containing KAWA buffer (section 2.1 / step 3). We use Qiagen Proteinase.K, 20 mg/ml (QIAGEN AG, Basel, Switzerland; Product code 19131).
Alternatively, a commercial kit may be used as mentioned in Section Materials, following the manufacturer’s protocols. It is recommended that the manufacturer’s guidelines are checked each time kits are ordered to ensure any updates/changes made since development of this SOP are incorporated.
2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
Add 100µL-200µL *) in Collection Microtubes (Qiagen).
*) KAWA Buffer: 10millimolar (mM), 1millimolar (mM), 0.5%, 50μg/ml, 8.0.
Preparation of 100 ml KAWA Buffer:
| A | B | C |
|---|---|---|
| Nr | Amount | Material |
| 1 | 0.121 g | Tris Base |
| 2 | adjust to pH 8.0 with 1M HCl (ca. 0.5 ml) | |
| 3 | 0.037 g | EDTA |
| 4 | 0.5 ml | Tween 20 |
| 5 | 5 mg (250 µl of Qiagen Proteinase.K, 20 mg/ml) | Proteinase K |
Aliquot in 1.5 ml tubes.
STORE AT -20°C.
Add stainless steel ball 3 mm.
Add tissue sample.
Homogenize on the Retsch Mixer Mill (Qiagen) for 2x3 min at 25 Hz, turning plate after the first period, then centrifuge.
Heat tubes at 95°C for 0h 20m 0s.
Centrifuge, transfer in 0.5 ml lowbind Eppendorf tubes.
Store frozen at -20°C (avoid freeze-thaw cycles, homogenates may be stored for several weeks at 4°C).
2.2 Reaction cleanup
Materials:
We use the MinElute Reaction Cleanup Kit of Qiagen (QIAGEN AG, Basel, Switzerland; Product code 28206).
A cleanup step using the MinElute Reaction Cleanup Kit of Qiagen (QIAGEN AG, Basel, Switzerland; Product code 28206) is recommended after the proteinase K extraction as this seems to reduce the variance in the number of sequencing reads between individual libraries in multiplex runs.
This step is not necessary if using a commercial nucleic acid extraction kit. However, a reverse transcription step is required if using an RNA extraction kit. For example, using the GenElute™ Total RNA Purification Kit from Sigma-Aldrich (Merck, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland; Product code: RNB100), the following cDNA production kit was successfully used: LunaScript® RT SuperMix Kit (Bioconcept AG, Allschwil, Switzerland; Product code NEB E3010S). Also, if RNA is extracted it may be beneficial to omit the DNAse step to maximize the yield of nucleic acids.
Procedure for reaction cleanup using the Qiagen MinElute Reaction Cleanup Kit:
Prepare MinElute column setup on 2ml collection tube.
Add 300µL to 75µL.
Load 375µL to column setup.
Centifuge the sample of last step for 0h 1m 0s.
Discard flow-through, re-assemble.
Add 750µL to column setup.
Centifuge the sample of last step for 0h 1m 0s.
Discard flow-through, re-assemble.
Centifuge the sample of last step for 0h 2m 0s.
Place MinElute column in NEW 1.5ml Eppendorf tube .
Add 12µL to centre of MinElute Column.
Incubate for 0h 1m 0s @ -20Room temperature.
Centrifuge for 0h 1m 0s.
2.3 DNA extract quantification
DNA extracted with a commercial kit or after clean-up may be quantified to assess the extraction process and enable normalisation of DNA concentration. One common method is to use a Nanodrop ND 1000 Spectrophotometer. DNA should be diluted to 10-50ng/μl using DNA-free water. Negative controls should read ~0 ng/μl.
Controls:
A negative extraction control (with no tissue) should be run in parallel with all batches of sample extraction and quantified alongside all tissue extractions
3 Whole Genome Amplification (WGA)
Materials:
We use the GenomePlex® Complete Whole Genome Amplification Kit WGA2 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland; Product code WGA2-50RXN).
Procedure for whole genome amplification using the Sigma GenomePlex® Complete Whole Genome Amplification Kit WGA2:
3.1 WGA Step 1: Fragmentation
Run Thermocycler program (Program: incubation at 95°C, runs 0h 30m 0s).
(To assure the Thermocycler is ready when needed.)
Use DNA/cDNA sample: Transfer 10µL of section 2.2/step 19 into new 8-Strip Microtubes .
Add 1µL to each DNA tube of previous step.
Heat for 0h 4m 0s @ 95°C in Thermocycler. Immediately cool . 95On ice.
3.2 WGA Step 2: Library Preparation
Add 2µL to DNA of previous step.
Add 1µL to DNA of previous step. Vortex and centrifuge.
Heat for 0h 2m 0s @ 95°C in Thermocycler. Immediately cool . 95On ice.
Add 1µL to DNA of previous step. Vortex and centrifuge.
Run Thermocycler program with WGA Library Prep Rxn.
Program:
incubation at 16°C, runs 0h 20m 0s;
incubation at 24°C, runs 0h 20m 0s;
incubation at 37°C, runs 0h 20m 0s;
incubation at 75°C, runs 0h 5m 0s;
cool to and hold at 4°C
3.3 WGA Step 3: Amplification
Add 48µL to each reaction tube of previous step (WGA Library Prep Rxn).
Add 7.5µL to each reaction tube of previous step.
Add 5µL to each reaction tube of previous step. Vortex and centrifuge.
Run Thermocycler program.
Program:
Initial incubation: 0h 3m 0s at 95°C;
17 cycles of 0h 0m 15s at 94°C; 0h 5m 0s at 65°C;
cool to and hold 4°C
Store short term 4°C, long term -20°C.
OPTIONAL: Check on gel: Run 5µL on 1.4% TBE gel, 4µL, 4µL at 70 V/cm for 0h 30m 0s.
3.4 WGA Step 4: Reaction Cleanup
Prepare MinElute column setup on 2ml collection tube.
Add 300µL to 75µL.
Load 375µL to column setup.
Centifuge the sample of last step for 0h 1m 0s.
Discard flow-through, re-assemble.
Add 750µL to column setup.
Centifuge the sample of last step for 0h 1m 0s.
Discard flow-through, re-assemble.
Centrifuge the sample of last step for 0h 2m 0s.
Place MinElute column in NEW 1.5ml Eppendorf tube .
Add 10µL to centre of MinElute Column.
Incubate for 0h 1m 0s @ -20Room temperature.
Centrifuge for 0h 1m 0s.
4 WGA Product Check by Gel Electrophoresis
Gel electrophoresis of DNA in an agarose gel is a standard technique in molecular biology, but equipment, reagents, staining and visualization varies considerably between laboratories, and according to local health & safety controls. Therefore, this SOP suggests general conditions that need to be adapted to each laboratory.
4.1 Make a 1.2% TBE agarose gel (1xTBE pH: 9.0) containing 0.0001% Ethidium Bromide*)
Place 80mL + 1g in a 500ml Erlenmeyer flask.
Heat in microwave at max intensity for 0h 2m 0s with intermittent interruption for shaking (take care not to overheat).
Add 8µL and cast the gel.
Wait 0h 30m 0s at -20Room temperature or store in the fridge at 4°C.
4.2 Gel loading and running
Prepare Size Standard (e.g. Thermo Scientific™ GeneRuler DNA Ladder Mix, ready-to-use; Order Nr. 10181070) and samples (whole genome amplification products) for gel loading by mixing 3µL with 3µL to be prepared as follows:
Loading Buffer Preparation for PCR Amplification Product Electrophoresis (10ml):
Add 1.5g to 10mL, adjust pH to 9.0.
Add 5mg (adjust amount visually, may be too high).
Carefully pipet the 6µL into individual wells of the gel, beginning with the Size Standard at the leftmost well.
Run at 70V for approximately 0h 30m 0s (depending on size of gel), ensuring the DNA does not run off the gel.
4.3 Visualization
Visualize your DNA fragments in UV light (with appropriate safety precautions); if the WGA reaction has been successful it shows as a smear of approximately 400 - 1000 base pairs in length. Your negative controls should not contain bands.
4.4 Recording
Keep a permanent record of your gel (electronic and/or hard copy) as proof that the WGA reaction was successful and contaminant free.
5 Sequencing Library Preparation
Materials:
The library for nanopore sequencing is produced with the Ligation Sequencing Kit SQK-LSK109 of Oxford Nanopore Technologies for sequencing on the flowcell type R.9.4.1 (flowcell ID: FLO-Min106D), following the manufacturer’s recommendations with some minor modifications.
5.1 Library Preparation Step 1: DNA End-Prep
Transfer 120 ng total DNA of section 3 into new 8-strip Microtubes .
Add MH2O to total 54µL.
Add 3.5µL.
Add 3µL.
Mix by flicking, spin down.
RUN Thermocycler program.
Program:
incubate 0h 30m 0s at 20°C/0h 20m 0s at 65°C,
transfer contents to 1.5ml Eppendorf tube.
Add 60µL (resuspended). Mix by flicking tube.
Incubate for 0h 10m 0s @ 4Room temperature on HULA mixer.
Spin and pellet on magnet until clear. Pipette off supernatant, keep on magnet.
Wash beads on magnet with 200µL. Pipette off supernatant, do not disturb pellet.
Wash beads on magnet with 200µL. Pipette off supernatant, do not disturb pellet.
On magnet, pipette off residual EtOH.
Dry for 0h 0m 30s.
Resuspend in 61µL.
Incubate for 0h 5m 0s at Room temperature
Pellet on magnet until clear.
Collect 61µL eluate. May store at 4°C 0h 5m 0s.
OPTIONAL: Quantify 1µL of product on QuBit. Note DNA concentration (ng/μl).
5.2 Library Preparation Step 2: Native Barcode Ligation
Add 22.5µL of eluted DNA of Endrepair product into new 1.5 ml Eppendorf tube; mix by pipetting. Use total 100fmol-200fmol (=ca. 35-60 ng).
Add 2.5µL to each reaction tube of previous step. Note Barcode Numbers!
Add 25µL to each reaction tube of previous step; mix by pipetting.
Incubate for 0h 10m 0s @4Room temperature.
Add 50µL (resuspended). Mix by flicking.
Incubate for 0h 5m 0s @4Room temperature on HULA mixer. Spin down.
Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
Wash beads on magnet with 200µL. Pipette off supernatant, do not disturb pellet.
Wash beads on magnet with 200µL. Pipette off supernatant, do not disturb pellet.
Remove residual by spin on magnet. Pipette off residual EtOH.
Dry for 0h 0m 30s.
Resuspend in 26µL.
Incubate for 0h 2m 0s @4Room temperature.
Pellet on magnet until clear.
Collect 26µL and transfer to 1.5ml Eppendorf tube.
MUST DO: Quantify 1µL of product on QuBit. Note DNA concentration (ng/μl).
Pool equimolar amounts of each barcoded sample to 1.5ml Eppendorf tube (to 100-200 fmol total (=ca. 60 ng)).
Dilute single pooled Barcode ligation product to 65µL.
5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
Use 60µL.
Add 25µL.
Add 10µL.
Add 5µL. Mix by flicking, spin down.
Incubate for 0h 10m 0s @4Room temperature.
Add 40µL. Mix by flicking
Incubate for 0h 15m 0s @ 4Room temperature on HULA mixer. Spin down.
Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
Wash beads with 250µL. Wait 0h 3m 0s on Magnet. Resuspend by flicking, pellet, remove supernatant.
Wash beads with 250µL. Wait 0h 3m 0s on Magnet. Resuspend by flicking, pellet, remove supernatant.
Remove residual by spin.
Dry 0h 0m 30s.
Resuspend in 15µL.
Incubate for 0h 10m 0s @ 37°C.
Pellet on magnet until clear.
Collect library (15µL) from previous step into new Eppendorf tube.
Quantify 1µL on QuBit. Use appropriate amount for 16 ng of library for next step; dilute with EB buffer.
6 Nanopore Sequencing
Priming and Loading the Flowcell
6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
Prepare Flowcell (perform QC on MinION). Record number of active pores.
Prepare the flow cell priming mix : Add 30µL directly to Flush Buffer (FB) tube. Mix.
Load flow cell with 800µL via priming port. Spot on closed!!!
Wait for 0h 5m 0s.
Prepare LIBRARY for loading: add 38µL to 1.5ml Eppendorf tube.
Add Library : Add 26µL to 1.5ml Eppendorf tube (mixed immediately before use).
Add Library : Add 12µL from section 5.3 to 1.5 ml Eppendorf tube.
Load flow cell with 200µL via priming port. Spot on closed!!!
Add 75µL from via SpotON sample port. Add drop by drop!
Close priming port, SpotOn port, perform sequencing on a MinION (Flongle, GridION, Promethion) using protocols.io method https://www.protocols.io/view/starting-a-minion-sequencing-run-using-minknow-7q6hmze; make sure to use flow cell type LSK109 and barcode kit EXP-NBD104 (option now available).
6.2 Nanopore Sequencing Step 2: Flow Cell Storage
Prepare wash mix: Add 20µL to 1.5ml Eppendorf tube.
Add 380µL to same 1.5ml Eppendorf tube. Vortex.
Open inlet port, add 400µL via priming port. Close priming port after loading (Spot on closed!!!).
Wait for 0h 30m 0s @37Room temperature.
Add 500µL via priming port. Close priming port after adding (Spot on closed!!!).
Remove spare contents in flow cell. Aspirate 1000µL from empty flow cell via trash removal port top left. Spot on closed!!!
Store in fridge.
7 Raw Data Processing and Analysis
For Raw Data Processing and Analysis, please see section "Guidelines".
