Aug 08, 2025
P. vivax microhaplotype deep sequencing assays
- Angela Rumaseb1,
- Trent Peter2,
- Ashley Osborne1,
- Hidayat Trimarsanto1,
- Mariana Kleinecke1,
- David Hawkes2,
- Sarah Auburn1
- 1Menzies School of Health Research;
- 2Australian Genome Research Facility
Protocol Citation: Angela Rumaseb, Trent Peter, Ashley Osborne, Hidayat Trimarsanto, Mariana Kleinecke, David Hawkes, Sarah Auburn 2025. P. vivax microhaplotype deep sequencing assays. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92boml3e/v1
Manuscript citation:
Kleinecke, M., Sutanto, E., Rumaseb, A. et al. Microhaplotype deep sequencing assays to capture Plasmodium vivax infection lineages. Nat Commun 16, 7192 (2025). https://doi.org/10.1038/s41467-025-62357-x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 04, 2024
Last Modified: August 08, 2025
Protocol Integer ID: 106922
Keywords: Plasmodium vivax, microhaplotype, rhAmpSeq, amplicon sequencing, illumina, plasmodium vivax marker, plasmodium species confirmation marker, deep sequencing assay, microhaplotype loci, australian genomic research facility, high diversity snp, putative drug resistance marker, characterization of infection diversity, infection diversity, throughput multiplex pcr, integrated dna technology
Funders Acknowledgements:
Bill & Melinda Gates Foundation
Grant ID: INV-043618
Abstract
This SOP is adapted from a high throughput rhAmpSeq library preparation protocol by integrated DNA technologies (IDT) and has been optimized at the Australian Genomic Research Facility. The SOP describes the laboratory procedures for amplifying a panel of 98 Plasmodium vivax markers, using high-throughput multiplex PCR, for downstream genotyping by sequencing on the Illumina platform. The 98 markers include a panel of 93 microhaplotype loci (loci comprising of 3 or more globally high diversity SNPs) distributed across the P. vivax genome, as well as one Plasmodium species confirmation marker and 4 putative drug resistance markers.
Further information on potential use cases for P. vivax microhaplotype genotyping, including recurrence classification, characterization of infection diversity, and spatiotemporal monitoring, can be found in Siegel et al [1].
Materials
| Item | Supplier | Size | Catalog# | |
| Reagents | ||||
| IDTE, pH 7.5 | IDT | 10 x 2 mL | 11-01-02-02 | |
| IDTE pH 8.0 | IDT | 10 x 2 mL | 11-01-02-05 | |
| Nuclease-Free Water | IDT | 10 x 2 mL | 11-04-02-01 | |
| rhAmpSeq Library Kit | IDT | 16 rxn 100 rxn 500 rxn 5000 rxn | 10000064 10000065 10000066 10000067 | |
| rhAmpSeq pv custom PCR panel | IDT | 0.4nmole (1.6mL) | Sales Order# 9413072 WO#3363760 | |
| xGen 10nt UDI Index Primer Plates | IDT | Plate 1-4 Plate 1-8 Plate 1-16 | 10008052 10008053 10008054 | |
| SPRIselect | Beckman Coulter | 5 mL 60 mL 450 mL | B23317 B23318 B23319 | |
| Ethanol (200 proof) | ||||
| High Sensivity D1000 ScreenTape | Agilent | 7 ScreenTapes | AG 5067-5584 | |
| High Sensitivity D1000 Reagents | Agilent | 112 reactions | AG 5067-5585 | |
| Collibri™ Library Quantification Kit | Invitrogen | A38524500 | ||
| Consumables | ||||
| DNA LoBind Tubes | Eppendorf | 250 pcs (1.5 mL) 250 pcs (2 mL) | 022431021 022431048 | |
| Eppendorf twin.tec‱ microbiology PCR Plate 96, semi-skirted, 250 µL | Eppendorf | 10 pcs | 0030129326 | |
| Axygen™ 8-Strip PCR Tubes, 0.2 mL | Thermo Fisher Scientific | 125 pcs | 14-222-251 | |
| Microseal 'B' PCR Plate Sealing Film | Bio-Rad | 100 pcs | MSB1001 | |
| Agilent Loading Tips | Agilent | 112 pcs | AG 5067-5598 | |
| Agilent Strip caps | Agilent | 120 pcs | AG 401425 | |
| Agilent Optical tube strip | Agilent | 120 pcs | AG 401428 | |
| Equipment | ||||
| DynaMag™2 Magnet or equivalent | Thermo Fisher Scientific | 12321D | ||
| Agilent 4150 TapeStation | Agilent | |||
| 96-well thermocycler | ||||
| Microcentrifuge | ||||
| Vortex | ||||
| Centrifuge with plate adapter |
Troubleshooting
Before start
The only safe stopping point in the procedure is after clean-up (Step 48).
Sample Preparation
Input DNA does not require prior quantification or normalization. Using a cox1 P. vivax qPCR assay (Gruenberg et al., 2018), input DNA with a CT value of <30 (roughly ≥100 parasites/μL) yielded a read depth of ≥100 reads/marker. If DNA amount is limited (11μL of DNA is required for this protocol), gDNA can be diluted using IDTE, pH 8.0.
Rebalanced Working Primer Preparation
Thaw an aliquot of Rebalanced rhAmpSeq PCR Panel (Forward and Reverse Stock Primer Pool from -20°C storage.
After thawing the primers, briefly vortex, then centrifuge.
Prepare working primer pools using a 1 in 5 dilution.
To sequence 96-samples, add 42μL of Forward/Reverse Stock Pool to 168μL of IDTE, pH 7.5.
Tip: Prepare multiple aliquots of rhAmpSeq forward/reverse primer pools and store at -20°C to avoid multiple freeze-thaw cycles.
Perform Targeted rhAmpSeq PCR_1
Thaw 4X rhAmpSeq Library Mix 1 and Rebalanced rhAmpSeq Working PCR Panel (Forward and Reverse Working Pool) completely to room temperature (15-20°C).
After thawing the reagents, briefly vortex and then centrifuge.
Prepare the following PCR_1 master mix in a Lo-bind Eppendorf tube for the number of samples being processed:
| Component | Per reaction | |
| 4X rhAmpSeq Library Mix 1 | 5μL | |
| rhAmpSeq pv custom PCR Panel – Forward Working Pool | 2μL | |
| rhAmpSeq pv custom PCR Panel – Reverse Working Pool | 9μL | |
| Total | 9μL |
Targeted rhAmp PCR_1 master mix
Briefly vortex and centrifuge down the mastermix.
In a new 96-well plate, add 9μL of PCR_1 master mix to each reaction well.
Add 11μL of gDNA to each reaction well.
Seal the plate, then briefly vortex and centrifuge.
Place the plate in a thermocycler and run the following program (Lid set to 105°C):
| Step | Cycle | Temperature | Duration | |
| Activate enzyme | 1 | 95°C | 10 min | |
| Amplify | 14 | 95°C | 15 sec | |
| 61°C | 8 min | |||
| Deactivate enzyme | 1 | 99.5°C | 15 min | |
| 4°C | Hold |
Targeted rhAmp PCR_1 Program
Once the program is complete, remove the plate from the thermocycler and proceed immediately to Dilute PCR_1 product.
Dilute PCR_1 product
Briefly vortex the PCR_1 product, then centrifuge.
In a new 96-well plate, add 95μL of Nuclease-Free Water to each well.
Transfer 5μL of the PCR_1 product to each well (To prepare a final dilution of 1 in 20 dilution). Pipette up and down to ensure the entire volume is dispensed.
Seal the plate and thoroughly vortex to mix, then centrifuge before proceeding immediately to Perform Indexing PCR_2.
Perform Indexing PCR
Thaw 4X rhAmpSeq Library Mix 2 and xGen 10nt UDI Index Primer Working Plate completely to room temperature (15-20°C).
Briefly vortex, then centrifuge the reagents.
Prepare the following PCR_2 master mix in a Lo-bind Eppendorf tube for the
number of samples being processed:
| Component | Per reaction | |
| 4x rhAmpSeq Library Mix 2 | 5μL | |
| Nuclease Free Water | 2μL | |
| Total | 7μL |
In a new 96-well plate, add 7 μL of PCR_2 master mix to each reaction well.
Add 2 μL of xGen 10nt UDI Index Primer Working Concentration to each corresponding well using a multichannel pipette.
Add 11μL of diluted rhAmpSeq PCR_1 product to each reaction well in a 96-well plate using a multichannel pipette.
Important! Note down the index plate and well position used for each sample.
Seal the plate, then briefly vortex and centrifuge.
Place the plate in a thermocycler and run the following program (lid set to 105°C):
| Step | Cycle | Temperature | Duration | |
| Activate enzyme | 1 | 95°C | 3 min | |
| Amplify | 24 | 95°C | 15 sec | |
| 60°C | 30 sec | |||
| 72°C | 30 sec | |||
| Final extension | 1 | 72°C | 1 min | |
| 4°C | Hold |
Indexing PCR Cycling Condition
Once the program is complete, remove the plate from the thermocycler and proceed immediately to Pool libraries.
Pool libraries
Using a multichannel pipette, pool each row by transferring 5μL of each indexed library into an 8-well strip tube (use 10μL if the total number of individual libraries processed is less than 20 samples).
Cap the strip tube, briefly vortex, then centrifuge down.
Combine all libraries from the strip tube into a single 2mL LoBind tube.
Important! Pool the complete volume from each well for an even sample coverage.
Properly seal and store the plate with remaining individual indexed libraries at -20°C then proceed immediately to clean up the pooled libraries.
Clean up library
Prepare 2mL of fresh 80% ethanol solution by combining 400μL of molecular-grade water and 1,600μL of molecular-grade ethanol (Absolute ethanol).
Transfer 100μL of the rhAmpSeq library pool to a new 1.5mL tube.
Vortex the SPRI beads thoroughly before use.
Add 70μL of SPRI (0.7X) to the library pool.
Thoroughly mix the contents of the tube by pipetting up and down.
Incubate for 10 minutes at room temperature.
Briefly pulse spin down the tube then place it on a magnetic rack for 5 minutes, or until the solution is clear.
Keeping the tube on the magnet, do the following:
Aspirate with the pipette, then discard the supernatant.
Add 1000μL of 80% ethanol to the tube.
Incubate at room temperature for 30 seconds.
Aspirate, then discard the supernatant.
Repeat the 80% ethanol wash one more time (steps 40.1 - 40.4).
Using a new P200 pipette tip, remove the remaining ethanol from the tube.
Allow the beads to dry for 3 minutes at room temperature.
Remove the tube from the magnet.
Add 22μL of IDTE, pH 8.0, to elute the library pool.
Thoroughly vortex to fully resuspend the beads, then briefly pulse spin down the tube.
Incubate at room temperature for 3 minutes.
Place the tube on the magnet to collect the beads for 1 minute, or until the solution is clear.
While keeping the tube on the magnet, transfer 20μL of the final library pool elution into a new 1.5mL LoBind tube, ensuring no beads are carried over.
Proceed to quantify and check the size of the library.
Store any remaining library pool at -20°C for up to 3 weeks.
Library QC
Quantify the library concentration using Illumina NGS library quantification kit (such as The Invitrogen Collibri Library Quantification Kit)
Using capillary electrophoresis, check the fragment size of the library before and after beads clean-up to ensure that adapter dimers have been successfully removed.
Sequencing
Follow Illumina's recommended protocol for a 2 x 150bp paired-end sequencing run on your Illumina platform
Protocol references
[1] Siegel, S.V., Trimarsanto, H., Amato, R. et al. Lineage-informative microhaplotypes for recurrence classification and spatio-temporal surveillance of Plasmodium vivax malaria parasites. Nat Commun 15, 6757 (2024). https://doi.org/10.1038/s41467-024-51015-3
[2] Gruenberg et al:
Plasmodium vivax molecular diagnostics in community surveys: pitfalls and
solutions. Malaria Journal (2018), 17:55.
https://doi.org/10.1186/s12936-018-2201-0
Acknowledgements
We thank the patients who contributed their samples to the study, and the health workers and field teams who assisted with the sample collections for use in the establishment in this assay. We acknowledge and thank the co-authors of the assay publication, named: Edwin Sutanto, Kian Soon Hoon, Paulo Manrique, Ernest Diez Benavente, Georgia Whitton, Sasha V. Siegel, Richard D. Pearson, Roberto Amato, Anjana Rai, Nguyen Thanh Thuy Nhien, Hoang Chau Nguyen, Ashenafi Assefa, Tamiru S. Degaga, Dagimawie Tadesse Abate, Awab Ghulam Rahim, Ayodhia Pitaloka Pasaribu, Inge Sutanto, Mohammad Shafiul Alam, Zuleima Pava, Tatiana Lopera-Mesa, Diego Echeverry, Tim William, Nicholas M. Anstey, Matthew J. Grigg, Nicholas P. Day, Nicholas J. White, Dominic P. Kwiatkowski, Aimee R. Taylor, Rintis Noviyanti, Daniel Neafsey, & Ric N. Price, for their contributions which supported the establishment of this assay. We thank the staff of the Australian Genome Research Facility (AGRF) for contributions to samples logistics, sequencing and informatics.