ARTIC-like Bacillus anthracis MLVA amplicon sequencing protocol for MinION
Ágnes Nagy, Gábor Tóth
Abstract
Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is one of the gold standard strain-level subtyping methods for outbreak-related Bacillus anthracis strains. The repeat numbers of 31 VNTR loci can be determined by capillary electrophoresis of PCR amplicons spanning repeat regions or in silico analysis of whole genome sequencing (WGS) data. However these methods require clear isolates of typeable strains and can be performed in fixed well-equipped high biosafety level laboratories.
We developed field-applicable amplicon sequencing protocol for Bacillus anthracis MLVA typing directly from environmental samples without isolating clear cultures of Bacillus anthracis strains. 62 primers were used for generating PCR amplicons for 31 Bacillus anthracis VNTR loci, according to MLVA31 typing scheme described by Beyer et al. 2012. The primers generating amplicons longer than 200 bp were used from MLVA31 typing scheme (44 primers for 22 VNTR loci). For amplicons shorter than 200 bp, primers were redesigned to generate longer amplicons (between 300-700 bp) suitable for MinION sequencing.
We optimized and tested this protocol on hungarian virulent Bacillus anthracis strains, a 34F2 Bacillus anthracis vaccine strain, and on spiked environmental samples in Hungarian Defence Forces field-deployable laboratory.
Before start
Isolate DNA from environmental samples suspected to contain Bacillus anthracis spores with Qiagen DNeasy Blood&Tissue kit or similar suitable for DNA isolation from Gram positive bacteria.
It is recommended to apply an extra mechanical lysis step (for ex. bead beating) before DNA isolation to increase the effectiveness of spore disruption.
Before MLVA analysis check the isolated DNA with Bacillus anthracis-specific real-time PCR assay for Bacillus anthracis DNA content.
Steps
Primer pool preparation
If required resuspend lyophilised primers at a concentration of 100µM each. Primer names, characteristics, concentrations and volumes required for primer stocks are listed in the table below.
Generate 500µL primer Pool 1 stock by adding 7µL, 13.5µL or 15.5µL of each primer to a 1.5mL Eppendorf labelled “Pool 1 (stock)”, following the table above.
Dilute primer Pool 1 stock 1:10 in molecular grade water, to generate Pool 1 working solution.
Generate 100µL primer Pool 2 stock by adding 5µL of each odd region primer to a 1.5mL Eppendorf labelled “Pool 2”, and adjust final volume to 100µL with molecular grade water.
Multiplex PCR
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2 mL 8-strip PCR tubes:
Component Pool 1 Pool 2
5X Q5 Reaction Buffer 5µL 5µL
10 mM dNTPs 0.5µL 0.5µL
Q5 Hot Start DNA Polymerase 0.25µL 0.25µL
BaMLVA Primer Pool 1 working solution or Pool 2 4.3µL 1.0µL
Nuclease-free water 12.45µL 15.75µL
Total 22.5µL 22.5µL
In the extraction and sample addition cabinet add 2.5µL DNA to each tube and mix well by pipetting.
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Set-up the following programs on a gradient thermal cycler, or a thermal cycler suitable for running 2 or more different PCR cycles in one time, or 2 thermal cyclers:
Program for Pool 1 PCR:
Step Temperature Time Cycles
Heat Activation 98°C 0h 0m 30s 1
Denaturation 98°C 0h 0m 15s 45
Annealing 65°C 0h 5m 30s 45
Hold 4°C Indefinite 1
Program for Pool 2 PCR:
Step Temperature Time Cycles
Heat Activation 98°C 0h 0m 30s 1
Denaturation 98°C 0h 0m 15s 45
Annealing 63°C 0h 5m 30s 45
Hold 4°C Indefinite 1
Equipment
| Value | Label |
|---|---|
| Veriti 96-Well Thermal Cycler | NAME |
| Applied Biosystems | BRAND |
| 4375786 | SKU |
Quantification and normalisation
Quantify 1µL PCR product using the Quantus Fluorometer using the ONE dsDNA assay.
DNA quantification using the Quantus fluorometer
Equipment
| Value | Label |
|---|---|
| Quantus | NAME |
| Fluorometer | TYPE |
| Promega | BRAND |
| E6150 | SKU |
Label a 1.5mL Eppendorf tube for each sample and assemble the following PCR dilution for each sample for final volume of 10µL:
Pool 1 PCR reaction volume (xµL) containing 196ng PCR amplicon
Pool 2 PCR reaction volume (xµL) containing 21ng PCR amlicon
Nuclease-free water volume (xµL) to a final volume of 10µL
Total amount of PCR amplicons per sample 217ng in 10µL
Dilute PCR amplicon pool of each sample 1:10 adding 90µLmolecular grade water, and mix well by pipetting.
Label a 0.2 mL PCR tube for each sample.
Use 10µL input for the One-pot native barcoding reaction to give a total of 21.7ng per sample.
Native barcoding
Barcode the amplicon pools using the one-pot native barcoding approach.
Set up the following reaction for each sample:
Component Volume
PCR dilution from previous step 10µL
Nuclease-free water 2.5µL
Ultra II End Prep Reaction Buffer 1.75µL
Ultra II End Prep Enzyme Mix 0.75µL
Total 15µL
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 500µL SFB and resuspend beads completely by pipette mixing.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Place on magnetic rack and incubate until the beads have pelleted and the supernatant is completely clear.
Remove supernatant and discard.
Pulse centrifuge and remove any residual SFB.
Bath the pellet in 500µLof room-temperature 75% volume ethanol without resuspending the beads.
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for 0h 5m 0sor until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).
Incubate at room temperature for 0h 10m 0s
Incubate at 65°C for 0h 10m 0s
Incubate on ice for 0h 1m 0s
Resuspend pellet in 30µL nuclease-free water, mix gently by either flicking or pipetting and incubate for 0h 5m 0s.
Place on magnetic rack and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
In a new 0.2 mL PCR tube set up the following reaction:
Component Volume
Previous reaction mixture 3.5µL
Nuclease-free water 3.7µL
NBXX barcode 2.5µL
Ultra II Ligation Master Mix 10µL
Ligation Enhancer 0.3µL
Total 20µL
Incubate at room temperature for 0h 20m 0s
Incubate at 65°C for 0h 15m 0s
Incubate on ice for 0h 1m 0s
In a new 1.5 ml Eppendorf tube pool all 20µL one-pot barcoding reactions together.
Add 1.8x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 216µL SPRI beads to 120µL 6-plex pooled one-pot native barcoding reactions.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for 0h 5m 0s at room temperature.
Place on magnetic rack and incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear.
Set up the following AMII adapter ligation and clean-up with SFB.
Set up the following AMII adapter ligation reaction:
Component Volume
Barcoded amplicon pool 30µL
NEBNext Quick Ligation Reaction Buffer (5X) 10µL
Adapter Mix (AMII) 5µL
Quick T4 DNA Ligase 5µL
Total 50µL
Place on magnetic rack and incubate until the beads have pelleted and the supernatant is completely clear.
Remove supernatant and discard.
Repeat step 15.8-15.11. to perform a second SFB wash.
Pulse centrifuge and remove any residual SFB.
Add 15µL EB and resuspend beads by pipette mixing.
Incubate at 37°C for 0h 8m 0s.
Place on magnetic rack and transfer final library to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
Incubate at room temperature for 0h 20m 0s.
Add 1x volume of SPRI beads to the sample tube (1:1 ratio of beads to sample volume) and mix gently by either flicking or pipetting. For example add 50µL SPRI beads to 50µL adapter ligation reaction.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for 0h 5m 0s at room temperature.
Place on magnetic rack and incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear.
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Add 200µL SFB and resuspend beads completely by pipette mixing.
Pulse centrifuge to collect all liquid at the bottom of the tube.
Quantify 1µLof the final library using the Quantus Fluorometer using the ONE dsDNA assay.
DNA quantification using the Quantus fluorometer
MinION sequencing
Prime the flowcell and load 20-25 ng sequencing library onto the flowcell.
Start the sequencing run using MinKNOW.
If required plug the MinION into the computer and wait for the MinION and flowcell to ben detected.
Choose flow cell 'FLO-MIN106' from the drop-down menu.
Then select the flowcell so a tick appears.
Click the 'New Experiment' button in the bottom left of the screen.
On the New experiment popup screen, select the running parameters for your experiment from the individual tabs:
Experiment: : Name the run in the experiment field, leave the sample field blank.
Kit: Selection: Select LSK109 and Native barcoding kit (EXP-NBD104 or EXP-NBD114 or EXP-NBD196).
Run Options: Set the run length to minimum 24 hours (you can stop the run once sufficient data has been collected as determined using RAMPART).
Basecalling: Leave basecalling turned but select 'superaccurate basecalling'.
Barcoding : Leave barcoding turned, turn on trim barcodes, but turn off Barcode both ends option.
Output: The number of files that MinKNOW will write to a single folder. By default this is set to 4000 but can be reduced to make RAMPART update more frequently.
Click 'Start run'.
Monitor the progress of the run using the MinKNOW interface.
