TITLE: PCR based amplicon sequencing of P. vivax antigens
AUTHORS: Paolo Bareng

[STEPS]
SECTION: Primer pool(s) preparation
1. Prepare the primer pools 1, 2, and 3 by reconstituting lyophilized primers to a concentration of 100µM using nuclease-free water
SECTION: Primer pool(s) preparation
1.1. 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.

 

 


 

 

 

 

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


 


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

 


 


 

 

SECTION: Multiplex PCR reaction
4. 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.

5. Transfer 5µL from each of the amplified pools into a new tube, then continue with the bead clean-up process.
SECTION: Bead clean up
6. Resuspend AMPure beads by vortexing. Then, add one volume of AMPure beads to the combined PCR product; mix by gentle flicking.
SECTION: Bead clean up
7. Incubate for 5 mins at room temperature, either on rotator, or gently flick every ~1 min.
SECTION: Bead clean up
8. Spin down the sample and pellet on a magnet until the eluate is clear and colourless
SECTION: Bead clean up
9. Leaving the tube on the magnet, carefully pipette off and discard the supernatant
SECTION: Bead clean up
10. 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.
SECTION: Bead clean up
11. Repeat ethanol wash (step 10)
SECTION: Bead clean up
12. 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)
SECTION: Bead clean up
13. 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).
SECTION: Bead clean up
14. Pellet beads on magnet until the eluate is clear and colourless.
SECTION: Bead clean up
15. Transfer eluate to fresh DNA LoBind tube. Retain 1 µl of DNA for quantification (yield should be >10ng/µL).
SECTION: Bead clean up
16. You can now discard the tube with beads.
SECTION: Indexing
17. Combine PCR components as follows. The reaction is for 1x sample.

 

 

SECTION: Indexing
18. Incubate the reaction into a thermal cycler following the conditions below.


 


 

SECTION: Indexing
19. Bead clean up. Refer to Bead clean up step.
SECTION: Pooling samples
20. 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.
SECTION: Pooling samples
21. 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.
SECTION: Pooling samples
22. Refer to Bead clean up step. 

 

 

SECTION: Pooling samples
23. Sample is now ready for library preparation and sequencing