PCR based amplicon sequencing of P. vivax antigens

Prepare the primer pools 1, 2, and 3 by reconstituting lyophilized primers to a concentration of 100µM using nuclease-free water

The tables below show the volume of each 100µM forward and reverse primers stock to be added in the respective pools. The total volume provided in the table is 100µL with a concentration of 5µM for each pool. However, it is recommended to make larger volume pool then split into aliquots.

A	B	C
AMA-1	1720	5
CSP	1712	5
CyRPA	1723	5
MSP8	1704	5
P41	1700	5
s12	1743	5
s28	1708	5
TRAMP	1747	5
TRAP	1731	5
Water	--	10
Total	--	100

A	B	C
DBP	4300	5
MSP9	4333	5
MSP1_1	4338	5
RBP1a_1	4318	5
RBP2a_1	4314	5
RBP2b_1	4357	5
RON2_1	4350	5
Water	--	30
Total	--	100

A	B	C
MSP1_2	4381	5
RBP1a_2	4387	5
RBP2a_2	4326	5
RBP2b_2	4303	5
RON_2	4310	5
Water	--	50
Total	--	100

Combine PCR components as follows. The reaction is for 1x sample. Volume and concentration are the same for all three pools.

A	B
2x KAPA HotStart HiFi RM	6.25
0.5µM primer pool (s)	1.5
Nuclease-free water	3.75
Template DNA (1-100ng)	1
Total	12.5

Incubate the reaction into a thermal cycler following the conditions below. Pools 1 and Pools 2/3 have different PCR conditions

A	B	C	D
Initial denaturation	95	3 mins.	1
Denaturation	98	20 secs.	35
Annealing	63	15 secs.
Extension	72	4 mins.
Final extension	72	5 mins.	1
Hold	4	∞	1

PCR conditions for Pool 1

A	B	C	D
Initial denaturation	95	3 mins.	1
Denaturation	98	20 secs.	35
Annealing	63	15 secs.
Extension	72	6 mins, 30 seconds
Final extension	72	10 mins.	1
Hold	4	∞	1

PCR conditions for Pools 2 and 3

Run 2-3 µL of the amplified product on a 1% agarose gel. Strong bands in the expected sizes indicate that the target genes have been amplified in the samples.

Transfer 5µL from each of the amplified pools into a new tube, then continue with the bead clean-up process.

Resuspend AMPure beads by vortexing. Then, add one volume of AMPure beads to the combined PCR product; mix by gentle flicking.

Incubate for 5 mins at room temperature, either on rotator, or gently flick every ~1 min.

Spin down the sample and pellet on a magnet until the eluate is clear and colourless

Leaving the tube on the magnet, carefully pipette off and discard the supernatant

Keep the tube on magnet, and wash beads with 200 µl of freshly prepared 75% ethanol (without disturbing the pellet; leave ~15 secs). Remove the 75% ethanol using a pipette and discard.

Repeat ethanol wash (step 10)

After the second ethanol wash, spin down the sample briefly then put the sample back on the magnet. Pipette off residual wash. Allow to dry (~1-2 min)

Remove the tube from the magnetic rack and resuspend pellet in 13 µl nuclease-free water by gentle flicking. Incubate for 5-10 minutes at 37 °C (heat block).

Pellet beads on magnet until the eluate is clear and colourless.

Transfer eluate to fresh DNA LoBind tube. Retain 1 µl of DNA for quantification (yield should be >10ng/µL).

You can now discard the tube with beads.

Combine PCR components as follows. The reaction is for 1x sample.

A	B
LongAmp mastermix	12.5
PCR Barcode (Nanopore)	1
Purified DNA	5-10
Nuclease-free water	1.5-6.5
Total	25

Incubate the reaction into a thermal cycler following the conditions below.

A	B	C	D
Initial denaturation	95	3 mins.	1
Denaturation	95	15 secs.	18
Annealing	62	15 secs.
Extension	65	5 mins., 30 secs.
Final extension	72	10 mins.	1
Hold	4	∞	1

Bead clean up. Refer to Bead clean up step.

Ensure that the concentration of each sample is equal when pooling multiple samples, targeting a total of 70-80 ng per sample. Calculate the necessary volume for each sample individually and then pool them together.

Total volume of pooled sample will be >100 µl, depending on the number of samples and volume added. Proceed to final bead clean-up before library sequencing preparation.

Refer to Bead clean up step.

Sample is now ready for library preparation and sequencing