Mar 04, 2025

Public workspacePf-SMARRTer- Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing v1.13

DOI
dx.doi.org/10.17504/protocols.io.e6nvwbwy7vmk/v1
  • 1University of North Carolina
  • IDEEL
Jonathan Juliano
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.e6nvwbwy7vmk/v1
Protocol Citation: Jonathan Juliano, Jacob Sadler 2025. Pf-SMARRTer- Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing v1.13. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbwy7vmk/v1
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: February 14, 2025
Last Modified: March 04, 2025
Protocol Integer ID: 123738
Keywords: Plasmodium falciparum, multiplex drug-resistance panel, PCR protocol
Abstract
This is a multiplex PCR protocol for amplifying ama1, crt, mdr1, dhfr, dhps, and k13. The PCR primers were adopted from:
  • AMA1 - PMID:29246158
  • CRT - designed for this protocol
  • MDR1 - PMID:15132750 (MDR184 designed for this protocol)
  • DHFR & DHPS - PMID:15273102 and Am J Trop Med Hyg. 2008 Jun; 78(6): 892–894
  • K13 – designed for this protocol
  • HeOME: Aranda-Díaz A, Vickers EN, Murie K, Palmer B, Hathaway N, Gerlovina I, et al. Sensitive and modular amplicon sequencing of diversity and resistance for research and public health. bioRxiv. 2024.

Schematic of Pf-SMARRTer Protocol



Guidelines
Infectious Disease Epidemiology and Ecology Lab University of North Carolina at Chapel Hill, Chapel Hill, NC, USA www.med.unc.edu/infdis/ideel

ABCD
Version Date Authors Notes
1.1 18-Mar-2022 K. Wamae - draft protocol, primer pooling and PCR optimisation
1.2 10-Jun-2022 K. Wamae - adjusted primer pool ratios
1.3 1-Jul-2022 K. Wamae - adjusted primer pool ratios - replaced K13-B primer (F2 for F)
1.4 2-Sept-2022 K. Wamae - renamed k13 primers across all document versions to indicate loci covered - changed the primer-pool ratios in section 1.2 back to those indicated in protocol version 1.2 since they performed better
1.5 19-December K.Wamae - Included expected amplicon lengths based on the 3D7 lab isolate
1.6 11-January 23 J. Juliano -Include new primers for mdr184, crt, k13 622I and HeOME - Add normalisation of P1 and P2 products into library prep
1.7 27-March 23 J. Juliano J. Bailey -Rebalancing of primers to try to normalise products - Updated primer location and size table
1.8 27-April 23 J. Juliano J. Bailey - Rebalance of primers
1.9 14-June 23 J. Juliano - Rebalance of primers
1.10 04-Nov-23 J. Sadler J. Juliano - Standardization of amplicon pooling - Removal of quantification before amplicon pooling
1.11 12-Feb-24 J. Juliano J. Sadler - Conversion to Nextera Prep - Addition of dhfr 164 and pfmdr1 1246 - Removal of pfmdr1 1034
1.12 05-14-24 J. Juliano J. Sadler C. Henelley - Increased PfCRT from 10uL fwd and rev primer → 15uL
1.13 07-02-24 J. Juliano J. Sadler A. Simkin -K13-F forward changed to address lack of coverage -K13-G forward changed to address lack of coverage


Materials
1.1 List of primers.
ABC
GenePrimer NamePrimer Sequence (5'-3')
ama1AMA1_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCATCAGGGAAATGTCCAGT
AMA1_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCCTGCATGTCTTGAACA
crtPFCRT_F_v2TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTGGAGGTTCTTGTCTTGG
PFCRT_R_v2GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTTGTGAGTTTCGGATGTTACA
mdr1MDR1_86_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTATGTGCTGTATTATCAGGAGGAAC
MDR1_86_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATTGTACTAAACCTATAGATACTAATGATAATATTATAGG
MDR1_184_F_v2TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGAGTTCAGGAATTGGTACGA
MDR1_184_R_v2GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCTCTTCTATAATGGACATGGT
MDR1_1246_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAAATGTAAATGAATTTTCAAACCAATCTGGA
MDR1_1246_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATAGCAGCAAACTTACTAACACGTTTAA
dhfrDHFR_51_59_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAGGTTTTTAATAACTACACATTTAGAGGTCT
DHFR_51_59_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCATTTACATTATCCACAGTTTCTTTGTT
DHFR_108/164_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAAATTACAAAATGTTGTAGTTATGGGAAGAA
DHFR_108/164_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTAAAAATTCTTGATAAACAACGGAACCT
dhpsDHPS_437_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAAATGATAAATGAAGGTGCTAGTGT
DHPS_437_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATACAGGTACTACTAAATCTCTTTCACTAATTTTT
DHPS_540_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATGCATAAAAGAGGAAATCCACAT
DHPS_540_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCGCAAATCCTAATCCAATATCAA
DHPS_581_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCGTTATAGGATACTATTTGATATTGGAT
DHPS_581_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGCAATAAATCTTTTTCTTGAATA
DHPS_613_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGATTAGGATTTGCGAAGAAAC
DHPS_613_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTGTGTATTTATTACAACATTTTGATCATTC
k13K13-A-432-466_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAAGTGAAGCCTTGTTGAAAGAAG
K13-A-432-466_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTACACATACGCCAGCATTGTTG
K13-B-461-531_F2TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGATGGTGTAGAATATTTAAATTCGATG
K13-B-461-531_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTACCATTTGACGTAACACCACA
K13-C-513-570_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTGAAACTGAGGTGTATGATCG
K13-C-513-570_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGATGATCTAGGGGTATTCAA
K13-F-567-630_F2TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCGAATGTAGAAGCATATGATCA
K13-F-567-630_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGTAATTAAAAGCTGCTCCTGA
K13-G-660-709_F2TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAAGCTAGAAGTTCAGGAGC
K13-G-660-709_RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCAAGCTGCCATTCATTTG
HeOMEHeOME-A F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTTAGTTTCGGTATTTTGTGTTCCTCTT
HeOME-A RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGAAATTTATCAGAGTTACAAAAGGGAAATC
HeOME-B F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTATCCTTATCATTATTTCCATCATTTTCTGG
HeOME-B RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAAATAAAAGGAACAATGTAATGGTTGAAAA
HeOME-C F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCCGAAAACATATCACTAGATCCAT
HeOME-C RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAAATTAAACATGATGCCACATTTTAGTAGT
HeOME-D F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCTATCATTACATGCTGACACAATAT
HeOME-D RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATTAGTTGTGGAGATGATAAAACTATCAAATT
HeOME-E F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATTTTCTTATATAACCTAAGTTGATGACTTGG
HeOME-E RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAACAGATGAAGTAACTACTCGATTAAATGA
HeOME-F F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCTTTTTCGTTGTATGTGCATAATCA
HeOME-F RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACATAATTCTAATGATATTGACCTTGTGCA
HeOME-G F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACATTCACACAAATAGAAAAATCTTCATTTTTC
HeOME-G RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATCAATAATCAAAATCATGATAACAACCAATTT
HeOME-H F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATAACTTAAATAAAAATATGGACGGCTCC
HeOME-H RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACATTCTTTCAATGCTTCCGAAA
1.2 Primer pool ratios

● Mix the primers above (both forward and reverse) into pools 1 and 2 in the volumes below.
ABCD
Primer Pool 1 volume Primer Pool 2 volume
AMA1 20 PFCRT_v2 18
MDR1-86 20 MDR1-184_v2 12
MDR1-1246 20 K13-B 18
K13-A 20 K13-F 12
K13-C 25 DHPS-540 24
K13-G 15 DHPS-613 18
DHPS-436-437 15 DHFR-51-59 24
DHPS-581 15 HeOME-A 24
DHFR-108/164 25 HeOME-C 18
HeOME-B 25 HeOME-E 18
HeOME-D 20 HeOME-G 36
HeOME-F 15 HeOME-H 12

Note
The volume listed for each target should be added for both forward and reverse primers. For example, AMA1 has a volume of 20µL listed. Add 20µL of both forward and reverse primer to the pool, 40µL total for the target if making one batch.

Table 1.3 - I7 index
ABCD
BeginningIndexEndTo order
CAAGCAGAAGACGGCATACGAGATCGCTCAGTTCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCGCTCAGTTCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTATCTGACCTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTATCTGACCTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTCGGATGTCGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTCGGATGTCGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCTTATGGAATGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCTTATGGAATGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTCCTATTGTGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTCCTATTGTGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGCGCGATGTTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGCGCGATGTTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAGAGCACTAGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAGAGCACTAGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGCCTTGATCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGCCTTGATCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCTACTCAGTCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCTACTCAGTCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTCGTCTGACTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTCGTCTGACTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGAACATACGGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGAACATACGGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCCTATGACTCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCCTATGACTCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTAATGGCAAGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTAATGGCAAGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGTGCCGCTTCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGTGCCGCTTCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCGGCAATGGAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCGGCAATGGAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGCCGTAACCGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGCCGTAACCGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAACCATTCTCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAACCATTCTCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGGTTGCCTCTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGGTTGCCTCTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCTAATGATGGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCTAATGATGGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTCGGCCTATCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTCGGCCTATCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAGTCAACCATGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAGTCAACCATGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGAGCGCAATAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGAGCGCAATAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAACAAGGCGTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAACAAGGCGTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGTATGTAGAAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGTATGTAGAAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTTCTATGGTTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTTCTATGGTTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCCTCGCAACCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCCTCGCAACCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGGATGCTTAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGGATGCTTAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATATGTCGTGGTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATATGTCGTGGTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAGAGTGCGGCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAGAGTGCGGCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGCCTGGTGGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGCCTGGTGGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGCGTGTCACGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGCGTGTCACGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCATACACTGTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCATACACTGTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCGTATAATCAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCGTATAATCAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTACGCGGCTGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTACGCGGCTGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGCGAGTTACCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGCGAGTTACCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTACGGCCGGTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTACGGCCGGTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGTCGATTACAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGTCGATTACAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCTGTCTGCACGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCTGTCTGCACGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATCAGCCGATTGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATCAGCCGATTGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGACTACATAGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGACTACATAGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATATTGCCGAGTGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATATTGCCGAGTGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGCCATTAGACGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGCCATTAGACGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATGGCGAGATGGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATGGCGAGATGGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTGGCTCGCAGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTGGCTCGCAGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTAGAATAACGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTAGAATAACGGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTCCATGTTGCGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTCCATGTTGCGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATTATCCAGGACGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATTATCCAGGACGTCTCGTGGGCTCGG
CAAGCAGAAGACGGCATACGAGATAGTGCCACTGGTCTCGTGGGCTCGGCAAGCAGAAGACGGCATACGAGATAGTGCCACTGGTCTCGTGGGCTCGG
Table 1.4 - I5 index
ABCD
BeginningIndexEndTo order
AATGATACGGCGACCACCGAGATCTACACTCGTGGAGCGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCGTGGAGCGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTACAAGATATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTACAAGATATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTACGTTCATTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTACGTTCATTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTGCCTGGTGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTGCCTGGTGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTCCATCCGAGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCCATCCGAGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGTCCACTTGTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGTCCACTTGTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTGGAACAGTATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTGGAACAGTATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCCTTGTTAATTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCCTTGTTAATTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGTTGATAGTGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGTTGATAGTGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACACCAGCGACATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACACCAGCGACATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCATACACTGTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCATACACTGTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGTGTGGCGCTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGTGTGGCGCTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACATCACGAAGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACATCACGAAGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCGGCTCTACTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCGGCTCTACTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGAATGCACGATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGAATGCACGATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACAAGACTATAGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACAAGACTATAGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTCGGCAGCAATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCGGCAGCAATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTAATGATGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTAATGATGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGGTTGCCTCTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGGTTGCCTCTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCGCACATGGCTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCGCACATGGCTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGGCCTGTCCTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGGCCTGTCCTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTGTGTTAGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTGTGTTAGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTAAGGAACGTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTAAGGAACGTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTAACTGTAATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTAACTGTAATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGGCGAGATGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGGCGAGATGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACAATAGAGCAATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACAATAGAGCAATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTCAATCCATTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCAATCCATTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTCGTATGCGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCGTATGCGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTCCGACCTCGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTCCGACCTCGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTTATGGAATTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTTATGGAATTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGCTTACGGACTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGCTTACGGACTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGAACATACGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGAACATACGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGTCGATTACATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGTCGATTACATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACACTAGCCGTGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACACTAGCCGTGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACAAGTTGGTGATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACAAGTTGGTGATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTGGCAATATTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTGGCAATATTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGATCACCGCGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGATCACCGCGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACTACCATCCGTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACTACCATCCGTTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGCTGTAGGAATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGCTGTAGGAATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCGCACTAATGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCGCACTAATGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGACAACTGAATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGACAACTGAATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACAGTGGTCAGGTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACAGTGGTCAGGTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCCATCTCGCCTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCCATCTCGCCTCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCTGCGAGCCATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCTGCGAGCCATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCGTTATTCTATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCGTTATTCTATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGCAACATGGATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGCAACATGGATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACGTCCTGGATATCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACGTCCTGGATATCGTCGGCAGCGTC
AATGATACGGCGACCACCGAGATCTACACCAGTGGCACTTCGTCGGCAGCGTCAATGATACGGCGACCACCGAGATCTACACCAGTGGCACTTCGTCGGCAGCGTC

Reagents


Consumables

  • Nuclease-free PCR microtubes/strips/plates (preferably LoBind).
  • Nuclease-free 1.5µL Eppendorf tubes (for master mixes, preferably LoBind).
  • Freezer blocks for microtubes and 1.5µL tubes to hold plates and tubes during processing.
  • P200 pipettor and tips
  • P20 pipettor and tips
  • 25µL reagent reservoir (3)
  • 96-well Flat-bottomed Assay Plates (Black)

Experiment set up

● Thaw reagents and sample DNA on ice.
● Clean pipettes and working bench tops with 70% ethanol.
● UV-treat the working surface, all equipment, and tubes/strips.
● Briefly vortex and centrifuge each reagent before use.



















Methods
Methods
Dilute the Concentration100 micromolar (µM) stock primers down to Concentration10 micromolar (µM) working solutions for each forward and reverse primer.

Prepare the two primer pools as indicated in section 1.1 [In materials] by mixing all forward and reverse primers in the ratios shown in section 1.2 [In materials].
Prepare the PCR master mix based on the table below.

  • Since there are two primer pools, the recipe below applies to both pools 1 and 2.
  • If samples are going to be sequenced in duplicate, this translates to translates to four PCR reactions per sample, i.e., two PCR replicates for primer pool 1 and two PCR replicates for primer pool 2.
  • Calculate the amount of reagents needed with a 10% excess to account for pipette loss.

AB
Reagents for a 25μl reaction 1x Reaction
Qiagen PCR Master Mix 12.5μl
Primer Pool+ 2.5μl
Template DNA* 2.0μl
PCR grade water 8.0μl
+The “primer pool mix” represents one for pool 1 that is separate from pool 2.
*Template DNA volume can be adjusted depending on the concentration of DNA by changing the amount of PCR grade water in the reaction.
All reagents, PCR master mix, PCR reagents and PCR mixes should be held in freezer blocks (tube or 96-well) unless stated otherwise.
Ensure that PCR reactions are labelled to be able to identify the sample name and which primer pool (A or B) is being used in the PCR.
Combine the master mix and sample DNA, seal caps/strips/plates, and place in a thermocycler using the following conditions:

ABCD
Step Duration Cycles Temperature
Initial activation step 15 min195° C
Denaturation Annealing Extension 30 sec 45 94° C
90 sec 60° C
90 sec 72° C
Final Extension 10 min172° C
HoldIndefinite14° C

Mixing of PCR products & 1:1 Speed-Bead purification
Mixing of PCR products & 1:1 Speed-Bead purification
50m 30s
50m 30s
Bring beads to TemperatureRoom temperature (Duration00:30:00 ).
30m
Temperature
In a 96 well LoBind PCR plate, combine the entire P1 and P2 PCR reaction into a single well creating the final multiplex amplicon pool.

  • Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
Vortex Speed-Beads thoroughly then add Amount50 µL to each well. Mix gently by pipetting.

Pipetting
Mix
Allow the mixture to incubate at TemperatureRoom temperature for Duration00:10:00 .

10m
Incubation
Temperature
If there are droplets on the sides of the wells following incubation, cover the plate with a foil seal and briefly spin the plate to avoid losing product.
Place the plate on a 96 well magnetic block and wait Duration00:02:00 -Duration00:03:00 for the solution to clear.

3m
Aspirate clear supernatant and discard.
Pipetting
Dispense Amount120 µL 80% Ethanol into each well, incubate Duration00:00:30 , then aspirate to wash.

30s
Incubation
Wash
Repeat for a total of 2 washes.
Aspirate final drops of EtOH with P20 pipette tips. Allow bead pellets to dry on the magnet for Duration00:01:00 -Duration00:03:00 .

4m
Remove Plate from magnet.

Mix
Dispense Amount25 µL of Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.

Pipetting
Mix
Cover plate with foil seal and briefly centrifuge to collect product in the bottom of wells.
Centrifigation
Place the plate on a 96 well magnetic block and wait Duration00:02:00 - Duration00:03:00 for the solution to clear.

3m
Transfer fluid to a new LoBind PCR plate for further processing.
Spot-quantitation
Spot-quantitation
Using Qubit dsDNA HS reagent, spot-quantify three samples to ensure amplicon DNA was not lost during purification following the manufacturer's protocol.
If needed, run the purified products on a fragment analyzer (e.g. TapeStation) to ensure that you have the desired PCR fragments (approx. 300-400bp) before proceeding to library preparation
Library Preparation v1.0 - Setup
Library Preparation v1.0 - Setup
Thaw Pre-Combined UDI 96-well plate, purified PCR reaction, and KAPA Master Mix.

Generation of UDI index plates.

Figure. Generation of UDI Combinatorial Index Plates. By ordering i5 (yellow) and i7 (blue) on plates in opposite orientation, UDI combinatorial plates of unique combinations of i5 and i7 indexes (yellow + blue= green) can be generated using a multichannel pipette to mix primers in a LoBind DNA plate. The column of i5 primers should be pipetted across all columns of the UDI Combinatorial plate and the i7 primer row pipetted across all rows of the plate.

The final concentration of the i5/i7 primer mix should be 10 micromolar. This can be done any number of ways, including diluting the 100 micromolar stock of each primer to 10 micromolar and then mixing.

  • UDI Combinatorial Plates can me made well ahead of time and frozen for use.
  • Typically we recommend making plates for a maximum of 10 indexing PCRs, thus if contamination should occur a new plate is accessed without wasting reagents.
Library Preparation v1.0 - Reaction Mixture
Library Preparation v1.0 - Reaction Mixture
Calculate the amount of reagents needed with a 10% excess to account for pipette loss.

Combine KAPA HiFi HotStart ReadyMix 2X and water in a tube then aliquot equal volumes into 0.2mL microtube strips. Pipette Amount17.0 µL Master Mix into each well with a multichannel pipettor.

Note
You may use reverse pipetting technique to avoid bubbles and expedite this step.

AB
ReagentVolume/Reaction
KAPA HiFi HotStart ReadyMix 2X 12.5 µL
Molecular Grade Water4.5 µL
Total 17.0 µL

Pipetting
Library Preparation v1.0 - Addition of Unique Dual-Indexes and PfSMARRT Amplicons
Library Preparation v1.0 - Addition of Unique Dual-Indexes and PfSMARRT Amplicons
Spin UDI plate to ensure there is no liquid on the plate sealing foil.

Remove the foil seal carefully to avoid contamination.
Add Amount2.5 µL of the appropriate UDI to each well. No need to mix yet.

Pipetting
Seal stock UDI plate before opening and adding samples to prevent contamination.
Add Amount5.0 µL Combined and Purified PfSMARRT amplicons. Gently mix by pipetting.

AB
ReagentVolume/Reaction
Master Mix (described above) 17.0 µL
UDI Primer 2.5 µL
Combined & Purified PfSMARRT DNA 5.0 µL
Total 24.5 µL

Pipetting
Mix
Library Preparation v1.0 - Cycling Conditions
Library Preparation v1.0 - Cycling Conditions
Program thermocycler with the following conditions for 8-12 cycles.

ABCD
StepDurationCyclesTemperature
Initial Activation Step3 min195° C
Denaturation 30 sec 8 cycles - 12 cycles95° C
Annealing 30 sec 55° C
Extension 30 sec 72° C
Final Extension5 min172° C
HoldIndefinite14° C
Note
There is no need to perform a PCR-clean up step after Library Preparation PCR. Proceed directly to quantification and pooling.

Library Preparation v1.0 - PicoGreen Quantification on Multimode Plate Reader
Library Preparation v1.0 - PicoGreen Quantification on Multimode Plate Reader
2m
2m
Quantify Amount2 µL of amplified libraries in 48µL of 1X PicoGreen reagent.

  • If reagent is not already provided at 1X, mix 1:200 dye to buffer to create a working solution.    
  •  If you are quantifying an entire 96-well plate of samples, a second plate will be necessary for assay standards.  
Pipetting
Add Amount2 µL of each sample to a black, flat bottomed polystyrene assay plate.  

  • The 90 degree angle where the well wall meets the flat bottom holds this droplet nicely.  

Pipetting
  • Add Amount2 µL of each standard to the appropriate wells.      
  • Standards 1-8 in duplicate=16 wells required.
Pipetting
Fill each sample and standard wells with 48µL 1X PicoGreen Reagent.

  • Pipette to mix thoroughly, careful not to introduce bubbles.  
Pipetting
Mix
 Cover plate with optical film and incubate for Duration00:02:00 at TemperatureRoom temperature before assaying.  

2m
Incubation
Temperature
Place the prepared quantitation plate into the multimode plate reader.

  • Plate reader should be set up to analyze Fluorescence Intensity (FI) with an excitation wavelength around 480nm and an emission wavelength around 580nm. 
Calculate each sample’s concentration by creating a standard curve and plotting unknown fluorescence values. Divide resulting value by two to report nanograms per microliter. 
To pool equimolar volumes, divide the total nanograms of DNA desired in the pool by the calculated DNA concentration for each sample,

For example:

  • A pool is needed containing 300 nanograms of each library.
  • The library DNA concentration is 64.25 ng/µL.
  • Volume of library to add to pool (x)=300/64.25 → x = 4.67µL

Nanograms per library can be adjusted based on sample concentration.
Lower sample concentrations will require lower total nanograms pooled.

Cleanup of final pooled libraries
Cleanup of final pooled libraries
59m 30s
59m 30s
Bring beads to TemperatureRoom temperature  (Duration00:30:00 ).   
                
  • Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
30m
Pipetting
Temperature
Vortex Speed-Beads thoroughly before use. 
Mix
In a LoBind 1.5mL tube, combine final pooled libraries with Speed-Beads at a 1:1 ratio.
Allow the mixture to incubate at TemperatureRoom temperature for Duration00:10:00 . 

10m
Incubation
Temperature
Gently spin tube to collect contents at the bottom.
Mix
Place the tube on a tube magnet and wait Duration00:02:00 -Duration00:03:00 for the solution to clear. 

5m
Aspirate clear supernatant and discard.
Keeping the tube on the magnet, dispense 80% Ethanol into the tube to wash. Ethanol wash volume should be higher than library and Speed-Bead mixture. 
Wash
Incubate Duration00:00:30 then aspirate to wash.

30s
Incubation
Wash
Repeat for total of 2 washes.
Wash
Aspirate final drops of EtOH with P20 pipette tips. Allow bead pellets to dry on the magnet for Duration00:01:00 -Duration00:03:00 .

  • Dry beads to satin-matte finish, but not to the point of cracking.
4m
Pipetting
Remove tube from magnet and dispense Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Pipetting
Mix
Allow the mixture to incubate for Duration00:05:00 .

5m
Incubation
Gently spin the tube to collect product in the bottom, then place on a tube magnet and wait Duration00:02:00 -Duration00:03:00 for the solution to clear.

5m
Mix
Remove fluid to a new LoBind tube. 
Quantify library pool using Qubit dsDNA HS reagent according to the manufacturers protocol.
The library pool is ready to prepare for sequencing.