Protocol: Single Nucleotide Polymorphism genotyping by High Resolution Melt Analysis
for Plasmodium vivax
This protocol describes the use of high resolution melt (HRM) analysis of 42-Single Nucleotide
Polymorphism (SNP) genotyping assays using whole amplicon melting for Plasmodium vivax
malaria. The SNP identified at each genotyping assay, together creates a 42-SNP barcode for P.
vivax that will identify individual parasite infections.
P. vivax 42-SNP Barcode
Assay
Ref.
Alt.
Chrom.
1
C
T
1
2
T
G
1
3
C
T
2
4
C
T
3
5
A
G
3
6
A
G
3
7
C
T
4
8
A
G
5
9 10 11
T G C
C A T
5 6 6
12
C
T
7
13
C
T
7
14
T
C
8
15
A
G
8
16
T
C
8
17
C
T
8
18
C
T
9
19
T
G
10
20
G
T
10
21
C
T
10
22
C
T
10
23
T
C
11
24
T
C
11
25
C
T
11
26
C
A
11
27
G
A
11
28
G
A
11
29
T
C
11
30
G
A
11
31
C
T
12
32
C
T
12
33
G
A
12
34
C
T
13
35
G
A
13
36
T
C
13
37
C
A
13
38
G
A
14
39
C
T
14
Method
1. Equipment
• Pipetters (2 μL, 20 μL, 200 μL, 1000 μL)
• Vortex
• Centrifuge
• HRM instrument
2. Consumables
• PCR plates
• Plate seals
• Pipettes
• Eppendorf tubes
3. Reagents
 2.5x Light Scanner Master Mix (BioFire Diagnostics, U.S.A. Catalog # HRLS-ASY-0003)
 Forward and reverse primers (see table for sequences)
 PCR-grade water (supplied in master mix kit)
 Control DNA to identify both the reference and alternate alleles for each assay
 Clinical samples
4. DNA Sample Preparation
Quantitate the concentration of total DNA in clinical samples based on OD260 using a
NanoDrop 3300 Fluorospectrometer (Thermo Scientific, U.S.A.). Alternatively, the genome
copy number of P. vivax DNA can be quantified in clinical samples by using the Primerdesign
P. vivax quantitative real-time PCR kit (primerdesign Catalog # Path-P.vivax standard)
according to manufacturer’s instructions.
If the amount of DNA sample is limited we recommend performing whole genome
amplification (WGA) on the clinical samples using the Illustra GenomiPhi V2 DNA
Amplification Kit (Catalog # 25-6600-30) according to the manufacturer's instructions.
Following WGA, we purified the DNA using Beckman Coulter Agencourt AMPure XP system
(Catalog # A63881) according to manufacturer’s instructions.
5. Experimental Set-up
The plate map must include controls for both the reference and alternate allele for each
assay. An example of a 48-well and 384-well plate map to test 6 clinical samples in duplicate
is shown on page 3.
1
40
T
C
14
41
A
G
14
42
C
A
14
6. Setting up the HRM plate and master mix
HRM Master Mix cocktail
Primer mix (For each assay: 0.1-0.5 μM of forward and reverse primers)*
PCR-grade water
2.5x Light Scanner Master Mix
DNA (1 ng/μl)
Total Volume
*See Table S2
1X (μL)
2
1
4
3
10
1.
Calculate the amount of HRM master mix needed by multiplying each component
(shown above for one reaction (1X)) by the number of PCR wells or test samples. Add
1X to account for volume loss.
2. Make primer mix (See Table S2) in 1X TE Buffer. A stock solution of this mixture can be
prepared and stored at 4 °C.
3. Prepare a dilution 1 ng/μl of DNA samples and controls in 1X TE Buffer. Note: if
background noise in the melting profile is observed the DNA may have contaminants
from the extraction method and a serial dilution of the DNA should be performed to
identify the optimal concentration of DNA to genotype your clinical sample.
4. Prepare the HRM master mix
5. Vortex and briefly centrifuge
6. Add 7 μL of the master mix to each well of the plate as defined on the plate map
7. Add 3 μL of DNA to each well as defined on the plate map and gently pipet to mix
8. Seal the plate with an optically clear pressure-sensitive adhesive film
9. Centrifuge at 1000 RPM for 1 minute
10. Place plate in HRM machine start program with defined HRM conditions (below)
HRM Conditions
Temperature
(°C)
95
94
64
95
55
95
Time
(sec)
120
30
60
15
15
15
40 cycles
(2-Step PCR)
HRM
7. Data Analysis
1. Adjust the pre- and post-melt normalization regions outside of the melting region
2. Define your genotypes as reference and alternate allele
3. Genotype samples by manual or automatic calling of SNP genotypes
2
48-well plate map
Assay 1 A
Assay 1 B
Assay 2 A
Assay 2 B
Assay 3 A
Assay 3 B
A
B
C
D
E
F
1
Control Ref.
Control Ref.
Control Ref.
Control Ref.
Control Ref.
Control Ref.
2
Control Alt
Control Alt
Control Alt
Control Alt
Control Alt
Control Alt
3
Sample 1
Sample 1
Sample 1
Sample 1
Sample 1
Sample 1
4
Sample 2
Sample 2
Sample 2
Sample 2
Sample 2
Sample 2
5
Sample 3
Sample 3
Sample 3
Sample 3
Sample 3
Sample 3
6
Sample 4
Sample 4
Sample 4
Sample 4
Sample 4
Sample 4
7
Sample 5
Sample 5
Sample 5
Sample 5
Sample 5
Sample 5
8
Sample 6
Sample 6
Sample 6
Sample 6
Sample 6
Sample 6
384-well plate map
Assay 1
1
A Control Ref.
B Control Ref.
C Control Alt
D Control Alt
E Sample 1
F Sample 1
G Sample 2
H Sample 2
I Sample 3
J Sample 3
K Sample 4
L Sample 4
M Sample 5
N Sample 5
O Sample 6
P Sample 6
Assay 2
2
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 3
3
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 4
4
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 5
5
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 6
6
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 7
7
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 8
8
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 9
9
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 10
10
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 11
11
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 12
12
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 13
13
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 14
14
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 15
15
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 16
16
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 17
17
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 18
18
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 19
19
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 20
20
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 21
21
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 22
22
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 23
23
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
Assay 24
24
Control Ref.
Control Ref.
Control Alt
Control Alt
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 4
Sample 4
Sample 5
Sample 5
Sample 6
Sample 6
3