TITLE: ARTIC-like Bacillus anthracis MLVA amplicon sequencing protocol for MinION
AUTHORS: Ágnes Nagy, Gábor Tóth

[DESCRIPTION]
 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]
SECTION: Primer pool preparation
1. 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.
  

SECTION: Primer pool preparation
2. Generate 500 µL primer Pool 1 stock by adding 7 µL, 13.5 µL or 15.5 µL of each primer to a 1.5 mL Eppendorf labelled “Pool 1 (stock)”, following the table above.

 

SECTION: Primer pool preparation
3. Dilute primer Pool 1 stock 1:10 in molecular grade water, to generate Pool 1 working solution.
SECTION: Primer pool preparation
4. Generate 100 µL primer Pool 2 stock by adding 5 µL of each odd region primer to a 1.5 mL Eppendorf labelled “Pool 2”, and adjust final volume to 100 µL with molecular grade water.

 

SECTION: Multiplex PCR
5. 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                                                5 µL         5 µL
10 mM dNTPs                                                              0.5 µL     0.5 µL 
Q5 Hot Start DNA Polymerase                                   0.25 µL   0.25 µL 
BaMLVA Primer Pool 1 working solution or Pool 2    4.3 µL     1.0 µL
Nuclease-free water                                                   12.45 µL 15.75 µL
Total                                                                             22.5 µL    22.5 µL

 

SECTION: Multiplex PCR
6. In the extraction and sample addition cabinet add 2.5 µL DNA to each tube and mix well by pipetting.

 

SECTION: Multiplex PCR
7. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Multiplex PCR
8. 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            30 s          1
Denaturation        98 °C            15 s          45
Annealing             65 °C            330 s          45
Hold                        4 °C             Indefinite                  1

Program for Pool 2 PCR:

Step                     Temperature         Time                    Cycles

Heat Activation    98 °C            30 s          1
Denaturation        98 °C            15 s          45
Annealing             63 °C            330 s          45
Hold                        4 °C             Indefinite                  1


 

SECTION: Quantification and normalisation
9. Quantify 1 µL PCR product using the Quantus Fluorometer using the ONE dsDNA assay.


 

 


SECTION: Quantification and normalisation
10. Label a 1.5 mL Eppendorf tube for each sample and assemble the following PCR dilution for each sample for final volume of 10 µL:


Pool 1 PCR reaction volume (x µL) containing 196 ng PCR amplicon
Pool 2 PCR reaction volume (x µL) containing 21 ng PCR amlicon
Nuclease-free water volume (x µL) to a final volume of 10 µL

Total amount of PCR amplicons per sample 217 ng in 10 µL

 

SECTION: Quantification and normalisation
11. Dilute PCR amplicon pool of each sample 1:10 adding 90 µLmolecular grade water, and mix well by pipetting.

SECTION: Quantification and normalisation
12. Label a 0.2 mL PCR tube for each sample.
SECTION: Quantification and normalisation
13. Use 10 µL input for the One-pot native barcoding reaction to give a total of 21.7 ng per sample.

 

SECTION: Native barcoding
14. Barcode the amplicon pools using the one-pot native barcoding approach.
 


SECTION: Native barcoding
14.1. Set up the following reaction for each sample:

Component                                     Volume

PCR dilution from previous step      10 µL
Nuclease-free water                        2.5 µL 
Ultra II End Prep Reaction Buffer    1.75 µL
Ultra II End Prep Enzyme Mix          0.75 µL
Total                                                 15 µL
SECTION: Native barcoding
14.2. Incubate at room temperature for 10 min
Incubate at 65 °C for 10 min
Incubate on ice for 1 min
SECTION: Native barcoding
14.3. In a new 0.2 mL PCR tube set up the following reaction:

Component                             Volume

Previous reaction mixture       3.5 µL 
Nuclease-free water              3.7 µL
NBXX barcode                         2.5 µL
Ultra II Ligation Master Mix    10 µL
Ligation Enhancer                   0.3 µL
Total                                        20 µL


 


SECTION: Native barcoding
14.4. Incubate at room temperature for 20 min 
Incubate at 65 °C for 15 min
Incubate on ice for 1 min

 

SECTION: Native barcoding
14.5. In a new 1.5 ml Eppendorf tube pool all 20 µL one-pot barcoding reactions together.

 

SECTION: Native barcoding
14.6. Add 1.8x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add 216 µL SPRI beads to 120 µL 6-plex pooled one-pot native barcoding reactions.

 

 

SECTION: Native barcoding
14.7. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Native barcoding
14.8. Incubate for 5 min at room temperature.
SECTION: Native barcoding
14.9. Place on magnetic rack and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear.
SECTION: Native barcoding
14.10. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: Native barcoding
14.11. Add 500 µL SFB and resuspend beads completely by pipette mixing.
SECTION: Native barcoding
14.12. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Native barcoding
14.13. Place on magnetic rack and incubate until the beads have pelleted and the supernatant is completely clear.
SECTION: Native barcoding
14.14. Remove supernatant and discard.
SECTION: Native barcoding
14.15. Pulse centrifuge and remove any residual SFB.
 

SECTION: Native barcoding
14.16. Bath the pellet in 500 µLof room-temperature 75% volume ethanol without resuspending the beads.

SECTION: Native barcoding
14.17. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
SECTION: Native barcoding
14.18. 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.
SECTION: Native barcoding
14.19. With the tube lid open incubate for 5 minor until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).


SECTION: Native barcoding
14.20. Resuspend pellet in 30 µL nuclease-free water, mix gently by either flicking or pipetting and incubate for 5 min.

SECTION: Native barcoding
14.21. Place on magnetic rack and transfer sample to a clean 1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.

 

SECTION: Native barcoding
15. Set up the following AMII adapter ligation and clean-up with SFB.

 

SECTION: Native barcoding
15.1. Set up the following AMII adapter ligation reaction:

Component                                                          Volume

Barcoded amplicon pool                                       30 µL
NEBNext Quick Ligation Reaction Buffer (5X)     10 µL
Adapter Mix (AMII)                                                 5 µL
Quick T4 DNA Ligase                                             5 µL
Total                                                                       50 µL

 

SECTION: Native barcoding
15.2. Incubate at room temperature for 20 min.

SECTION: Native barcoding
15.3. 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 50 µL SPRI beads to 50 µL adapter ligation reaction.

 

SECTION: Native barcoding
15.4. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Native barcoding
15.5. Incubate for 5 min at room temperature.

SECTION: Native barcoding
15.6. Place on magnetic rack and incubate for 2 min or until the beads have pelleted and the supernatant is completely clear.

SECTION: Native barcoding
15.7. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: Native barcoding
15.8. Add 200 µL SFB and resuspend beads completely by pipette mixing.
 


SECTION: Native barcoding
15.9. Pulse centrifuge to collect all liquid at the bottom of the tube.
SECTION: Native barcoding
15.10. Place on magnetic rack and incubate until the beads have pelleted and the supernatant is completely clear.
SECTION: Native barcoding
15.11. Remove supernatant and discard.
SECTION: Native barcoding
15.12. Repeat step 15.8-15.11. to perform a second SFB wash.
SECTION: Native barcoding
15.13. Pulse centrifuge and remove any residual SFB.
 

SECTION: Native barcoding
15.14. Add 15 µL EB and resuspend beads by pipette mixing.

SECTION: Native barcoding
15.15. Incubate at 37 °C for 8 min.

 

SECTION: Native barcoding
15.16. Place on magnetic rack and transfer final library to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
SECTION: Native barcoding
16. Quantify 1 µLof the final library using the Quantus Fluorometer using the ONE dsDNA assay.
 

 


SECTION: MinION sequencing
17. Prime the flowcell and load 20-25 ng sequencing library onto the flowcell.
 

SECTION: MinION sequencing
18. Start the sequencing run using MinKNOW.


SECTION: MinION sequencing
18.1. If required plug the MinION into the computer and wait for the MinION and flowcell to ben detected.
SECTION: MinION sequencing
18.2. Choose flow cell 'FLO-MIN106' from the drop-down menu.
SECTION: MinION sequencing
18.3. Then select the flowcell so a tick appears.
SECTION: MinION sequencing
18.4. Click the 'New Experiment' button in the bottom left of the screen.
SECTION: MinION sequencing
18.5. 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'.
 

SECTION: MinION sequencing
18.6. Monitor the progress of the run using the MinKNOW interface.