Jul 15, 2025
Version 2
PfSMARRTer- Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (PfSMARRT v1.13) V.2
- Jonathan Juliano1,
- Jacob Sadler1
- 1University of North Carolina
- IDEEL
Protocol Citation: Jonathan Juliano, Jacob Sadler 2025. PfSMARRTer- Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (PfSMARRT v1.13). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbwy7vmk/v2Version created by Infectious Disease Epidemiology and Ecology Lab
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: June 25, 2025
Last Modified: July 15, 2025
Protocol Integer ID: 221045
Keywords: Plasmodium falciparum, multiplex drug-resistance panel, PCR protocol, Multiplex amplicon sequencing, malaria, antimalarial resistance, diversity, plasmodium falciparum streamlined multiplex antimalarial resistance, streamlined multiplex antimalarial resistance, plasmodium, biorxiv, pcr primer, protocol mdr1, mdr1, multiplex pcr protocol, pcr, version of pfsmarrt, modular amplicon
Funders Acknowledgements:
NIAID
Grant ID: R01AI156267
Abstract
PfSMARRTer- An updated version of PfSMARRT
This is a multiplex PCR protocol for amplifying ama1, crt, mdr1, dhfr, dhps, and k13. The PCR primers were adopted from:
- CRT - designed for this protocol
- 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
Image Attribution
Image logo generated by CharGTP. Protocol images generated un Biorender through UNC-CH license.
Guidelines
Infectious Disease Epidemiology and Ecology Lab University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
www.med.unc.edu/infdis/ideel
| A | B | C | D | |
| 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.
| A | B | C | |
| Gene | Primer Name | Primer Sequence (5'-3') | |
| ama1 | AMA1_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCATCAGGGAAATGTCCAGT | |
| AMA1_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCCTGCATGTCTTGAACA | ||
| crt | PFCRT_F_v2 | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTGGAGGTTCTTGTCTTGG | |
| PFCRT_R_v2 | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTTGTGAGTTTCGGATGTTACA | ||
| mdr1 | MDR1_86_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTATGTGCTGTATTATCAGGAGGAAC | |
| MDR1_86_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATTGTACTAAACCTATAGATACTAATGATAATATTATAGG | ||
| MDR1_184_F_v2 | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGAGTTCAGGAATTGGTACGA | ||
| MDR1_184_R_v2 | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCTCTTCTATAATGGACATGGT | ||
| MDR1_1246_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAAATGTAAATGAATTTTCAAACCAATCTGGA | ||
| MDR1_1246_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATAGCAGCAAACTTACTAACACGTTTAA | ||
| dhfr | DHFR_51_59_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAGGTTTTTAATAACTACACATTTAGAGGTCT | |
| DHFR_51_59_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCATTTACATTATCCACAGTTTCTTTGTT | ||
| DHFR_108/164_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAAATTACAAAATGTTGTAGTTATGGGAAGAA | ||
| DHFR_108/164_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTAAAAATTCTTGATAAACAACGGAACCT | ||
| dhps | DHPS_437_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAAATGATAAATGAAGGTGCTAGTGT | |
| DHPS_437_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATACAGGTACTACTAAATCTCTTTCACTAATTTTT | ||
| DHPS_540_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATGCATAAAAGAGGAAATCCACAT | ||
| DHPS_540_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCGCAAATCCTAATCCAATATCAA | ||
| DHPS_581_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCGTTATAGGATACTATTTGATATTGGAT | ||
| DHPS_581_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGCAATAAATCTTTTTCTTGAATA | ||
| DHPS_613_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGATTAGGATTTGCGAAGAAAC | ||
| DHPS_613_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTGTGTATTTATTACAACATTTTGATCATTC | ||
| k13 | K13-A-432-466_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAAGTGAAGCCTTGTTGAAAGAAG | |
| K13-A-432-466_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTACACATACGCCAGCATTGTTG | ||
| K13-B-461-531_F2 | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGATGGTGTAGAATATTTAAATTCGATG | ||
| K13-B-461-531_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTACCATTTGACGTAACACCACA | ||
| K13-C-513-570_F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTGAAACTGAGGTGTATGATCG | ||
| K13-C-513-570_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGATGATCTAGGGGTATTCAA | ||
| K13-F-567-630_F2 | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCGAATGTAGAAGCATATGATCA | ||
| K13-F-567-630_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGTAATTAAAAGCTGCTCCTGA | ||
| K13-G-660-709_F2 | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAAGCTAGAAGTTCAGGAGC | ||
| K13-G-660-709_R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCAAGCTGCCATTCATTTG | ||
| HeOME | HeOME-A F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTTAGTTTCGGTATTTTGTGTTCCTCTT | |
| HeOME-A R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGAAATTTATCAGAGTTACAAAAGGGAAATC | ||
| HeOME-B F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTATCCTTATCATTATTTCCATCATTTTCTGG | ||
| HeOME-B R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAAATAAAAGGAACAATGTAATGGTTGAAAA | ||
| HeOME-C F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCCGAAAACATATCACTAGATCCAT | ||
| HeOME-C R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAAATTAAACATGATGCCACATTTTAGTAGT | ||
| HeOME-D F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCTATCATTACATGCTGACACAATAT | ||
| HeOME-D R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATTAGTTGTGGAGATGATAAAACTATCAAATT | ||
| HeOME-E F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATTTTCTTATATAACCTAAGTTGATGACTTGG | ||
| HeOME-E R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAACAGATGAAGTAACTACTCGATTAAATGA | ||
| HeOME-F F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCTTTTTCGTTGTATGTGCATAATCA | ||
| HeOME-F R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACATAATTCTAATGATATTGACCTTGTGCA | ||
| HeOME-G F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACATTCACACAAATAGAAAAATCTTCATTTTTC | ||
| HeOME-G R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATCAATAATCAAAATCATGATAACAACCAATTT | ||
| HeOME-H F | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATAACTTAAATAAAAATATGGACGGCTCC | ||
| HeOME-H R | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACATTCTTTCAATGCTTCCGAAA |
1.2 Primer pool ratios
● Mix the primers above (both forward and reverse) into pools 1 and 2 in the volumes below.
| A | B | C | D | |
| 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
| A | B | C | D | |
| Beginning | Index | End | To order | |
| CAAGCAGAAGACGGCATACGAGAT | CGCTCAGTTC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCGCTCAGTTCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TATCTGACCT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTATCTGACCTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TCGGATGTCG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTCGGATGTCGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CTTATGGAAT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCTTATGGAATGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TCCTATTGTG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTCCTATTGTGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GCGCGATGTT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGCGCGATGTTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AGAGCACTAG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAGAGCACTAGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGCCTTGATC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGCCTTGATCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CTACTCAGTC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCTACTCAGTCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TCGTCTGACT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTCGTCTGACTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GAACATACGG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGAACATACGGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CCTATGACTC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCCTATGACTCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TAATGGCAAG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTAATGGCAAGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GTGCCGCTTC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGTGCCGCTTCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CGGCAATGGA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCGGCAATGGAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GCCGTAACCG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGCCGTAACCGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AACCATTCTC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAACCATTCTCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GGTTGCCTCT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGGTTGCCTCTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CTAATGATGG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCTAATGATGGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TCGGCCTATC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTCGGCCTATCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AGTCAACCAT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAGTCAACCATGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GAGCGCAATA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGAGCGCAATAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AACAAGGCGT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAACAAGGCGTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GTATGTAGAA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGTATGTAGAAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TTCTATGGTT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTTCTATGGTTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CCTCGCAACC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCCTCGCAACCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGGATGCTTA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGGATGCTTAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | ATGTCGTGGT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATATGTCGTGGTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AGAGTGCGGC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAGAGTGCGGCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGCCTGGTGG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGCCTGGTGGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGCGTGTCAC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGCGTGTCACGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CATACACTGT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCATACACTGTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CGTATAATCA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCGTATAATCAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TACGCGGCTG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTACGCGGCTGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GCGAGTTACC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGCGAGTTACCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TACGGCCGGT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTACGGCCGGTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GTCGATTACA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGTCGATTACAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CTGTCTGCAC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCTGTCTGCACGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | CAGCCGATTG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATCAGCCGATTGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGACTACATA | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGACTACATAGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | ATTGCCGAGT | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATATTGCCGAGTGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GCCATTAGAC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGCCATTAGACGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | GGCGAGATGG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATGGCGAGATGGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TGGCTCGCAG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTGGCTCGCAGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TAGAATAACG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTAGAATAACGGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TCCATGTTGC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTCCATGTTGCGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | TATCCAGGAC | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATTATCCAGGACGTCTCGTGGGCTCGG | |
| CAAGCAGAAGACGGCATACGAGAT | AGTGCCACTG | GTCTCGTGGGCTCGG | CAAGCAGAAGACGGCATACGAGATAGTGCCACTGGTCTCGTGGGCTCGG |
Table 1.4 - I5 index
| A | B | C | D | |
| Beginning | Index | End | To order | |
| AATGATACGGCGACCACCGAGATCTACAC | TCGTGGAGCG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCGTGGAGCGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTACAAGATA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTACAAGATATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TACGTTCATT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTACGTTCATTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TGCCTGGTGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTGCCTGGTGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TCCATCCGAG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCCATCCGAGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GTCCACTTGT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGTCCACTTGTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TGGAACAGTA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTGGAACAGTATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CCTTGTTAAT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCCTTGTTAATTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GTTGATAGTG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGTTGATAGTGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | ACCAGCGACA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACACCAGCGACATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CATACACTGT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCATACACTGTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GTGTGGCGCT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGTGTGGCGCTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | ATCACGAAGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACATCACGAAGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CGGCTCTACT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCGGCTCTACTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GAATGCACGA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGAATGCACGATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | AAGACTATAG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACAAGACTATAGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TCGGCAGCAA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCGGCAGCAATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTAATGATGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTAATGATGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GGTTGCCTCT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGGTTGCCTCTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CGCACATGGC | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCGCACATGGCTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GGCCTGTCCT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGGCCTGTCCTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTGTGTTAGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTGTGTTAGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TAAGGAACGT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTAAGGAACGTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTAACTGTAA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTAACTGTAATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GGCGAGATGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGGCGAGATGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | AATAGAGCAA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACAATAGAGCAATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TCAATCCATT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCAATCCATTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TCGTATGCGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCGTATGCGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TCCGACCTCG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTCCGACCTCGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTTATGGAAT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTTATGGAATTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GCTTACGGAC | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGCTTACGGACTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GAACATACGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGAACATACGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GTCGATTACA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGTCGATTACATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | ACTAGCCGTG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACACTAGCCGTGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | AAGTTGGTGA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACAAGTTGGTGATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TGGCAATATT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTGGCAATATTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GATCACCGCG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGATCACCGCGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | TACCATCCGT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACTACCATCCGTTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GCTGTAGGAA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGCTGTAGGAATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CGCACTAATG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCGCACTAATGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GACAACTGAA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGACAACTGAATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | AGTGGTCAGG | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACAGTGGTCAGGTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CCATCTCGCC | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCCATCTCGCCTCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CTGCGAGCCA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCTGCGAGCCATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CGTTATTCTA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCGTTATTCTATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GCAACATGGA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGCAACATGGATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | GTCCTGGATA | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACGTCCTGGATATCGTCGGCAGCGTC | |
| AATGATACGGCGACCACCGAGATCTACAC | CAGTGGCACT | TCGTCGGCAGCGTC | AATGATACGGCGACCACCGAGATCTACACCAGTGGCACTTCGTCGGCAGCGTC |
Reagents
- 80% Ethanol
- Elution Buffer
- Kapa HiFi Hotstart ReadymixKapa BiosystemsCatalog #KK2601
- Molecular Grade Water
- Pre-Combined UDI 96-well plate at 10µM total concentration (5µM each index, 10µM total concentration)
- 2X QIAGEN Multiplex PCR Master MixQiagenCatalog #206143 or QIAGEN Multiplex PCR KitQiagenCatalog #206145
- Quant-iT dsDNA Pico Green assay kit (Invitrogen)Life TechnologiesCatalog #P7589
- Invitrogen™ Qubit™ dsDNA Quantification Assay KitsFisher ScientificCatalog #Q32850
- Optional: If needed, fragment analyzer supplies (such as https://www.agilent.com/en/product/automated-electrophoresis/tapestation-systems/tapestation-dna-screentape-reagents/dna-screentape-analysis-228260)
- Optional: Commercial Illumina Library Quantification Kit with standards
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.
Troubleshooting
Methods
Dilute the 100 micromolar (µM) stock primers down to 10 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.
| A | B | |
| Reagents for a 25μl reaction | 1x Reaction | |
| Qiagen PCR Master Mix | 12.5μL | |
| Primer Pool+ | 2.5μL | |
| Template DNA* | 2.0-5.0μL | |
| PCR grade water^ | 5.0-8.0μL |
+The “Primer Pool” represents one of two pools in separate reactions.
*Template DNA volume can be adjusted depending on the concentration of DNA by changing the amount of PCR grade water in the reaction.
^ Amount of water varies inversely with Template DNA volume for a total of 10ul
All reagents, PCR master mix, primer pools, and samples 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 therein and which primer pool (A or B) is being used in the PCR.
All assays are carried out with positive and negative controls (preferably 2 of each). For a positive control, we use a mocked dried blood spot containing an estimated parasite density of 100 Pf/µL. The positive control you use should be extracted the same way as samples to be tested. For non-template control, we recommend using human DNA at a clinically relevant concentration (e.g. 16.77 ng/). If human DNA is not available, water can be used as a non-template control.
Controls should be run through the entire library prep process and checked in Step 30.
Combine the master mix and sample DNA, seal caps/strips/plates, and place the reaction in a thermal cycler using the following conditions:
| A | B | C | D | |
| Step | Duration | Cycles | Temperature | |
| Initial activation step | 15 min | 1 | 95° C | |
| Denaturation Annealing Extension | 30 sec | 45 | 94° C | |
| 90 sec | 60° C | |||
| 90 sec | 72° C | |||
| Final Extension | 10 min | 1 | 72° C | |
| Hold | Indefinite | 1 | 4° C |
Mixing of PCR products & 1:1 Speed-Bead purification
48m
Bring Speed-Beads to Room temperature (00:30:00 ).
30m
In a 96-well LoBind PCR plate, combine the entire P1 and P2 PCR reaction volume 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 50 µL to each well. Mix gently by pipetting.
Allow the mixture to incubate at Room temperature for 00:10:00 .
10m
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 00:02:00 -00:03:00 for the solution to clear.
3m
Aspirate the clear supernatant and discard.
Dispense 120 µL 80% Ethanol into each well, incubate 00:00:30 , then aspirate to wash.
30s
Repeat for a total of 2 washes.
Aspirate final drops of Ethanol with P20 pipette tips. Allow bead pellets to dry momentarily 00:00:30 -00:01:00 .
1m 30s
Remove plate from the magnet.
Dispense 25 µL of Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Cover plate with foil seal and briefly centrifuge to collect product in the bottom of wells before storing or proceeding to library preparation. The beads can remain in the eluant and may be added to the library preparation reaction. The eluant is not transferred to a new plate to limit cross-contamination of concentrated amplicon products.
Spot-quantitation
Using Qubit dsDNA HS reagent according to manufacturer instructions, spot-quantify three samples to ensure amplified DNA was not lost during purification.
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
Thaw Pre-Combined UDI 96-well plate, purified PCR reaction, and KAPA Master Mix.
Generation of UDI plates.
Figure. Generation of UDI (Unique Dual Index) plates. By ordering i5 (yellow) and i7 (blue) on plates in opposite orientation, UDI plates of combined i5 and i7 indexes (yellow + blue= green) can be made 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 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
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 17.0 µL Master Mix into each well with a multichannel pipettor.
Note
You may use reverse pipetting technique to avoid bubbles and expedite this step.
| A | B | |
| Reagent | Volume/Reaction | |
| KAPA HiFi HotStart ReadyMix 2X | 12.5 µL | |
| Molecular Grade Water | 4.5 µL | |
| Total | 17.0 µL |
Library Preparation v1.0 - Addition of Unique Dual-Indexes and PfSMARRTer Amplicons
Spin UDI plate to ensure there is no liquid on the plate sealing foil.
Remove the foil seal carefully to avoid contamination.
Add 2.5 µL of the appropriate UDI to each well. No need to mix yet.
Seal stock UDI plate before opening and adding samples to prevent contamination.
Add 5.0 µL Combined and Purified PfSMARRTer amplicons. Gently mix by pipetting.
| A | B | |
| Reagent | Volume/Reaction | |
| Master Mix (described above) | 17.0 µL | |
| UDI Primer | 2.5 µL | |
| Combined & Purified PfSMARRT amplicons | 5.0 µL | |
| Total | 24.5 µL |
Library Preparation v1.0 - Cycling Conditions
Program thermocycler with the following conditions for 8-12 cycles.
| A | B | C | D | |
| Step | Duration | Cycles | Temperature | |
| Initial Activation Step | 3 min | 1 | 95° C | |
| Denaturation | 30 sec | 8 - 12 | 95° C | |
| Annealing | 30 sec | 55° C | ||
| Extension | 30 sec | 72° C | ||
| Final Extension | 5 min | 1 | 72° C | |
| Hold | Indefinite | 1 | 4° C |
Check for contamination
Prior to proceeding to sample pooling into a final library, the libraries generated from positive and non-template controls should be checked for contamination.
It is expected that negative controls will have a detectable band on gel below the bands consistent with amplification products. Non-template control bands representing primer dimer that should yield data when processed through bioinformatic analysis (See 30.2).
Gel electrophoresis showing positive control [DNA extracted from 1,000 Pf/uL dried blood spot using Chelex-Tween (first lane)] and negative controls (second and third lane). The gel was made with 1% agarose and 1X TBE buffer then run at 100 volts for 45 minutes. The 100bp ladder's (lane 4) smallest fragment is 100bp, and the bright band being 500 bp. (Promega 100bp Ladder)
If the negative controls have a bands consistent with target amplification bands, the plate/samples should be repeated and the source of contamination investigated.
Options for final pooling
Option A: Pooling Based on qPCR Quantification
Bring Speed-Beads to Room temperature (00:30:00 ).
- Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
Vortex Speed-Beads thoroughly before use.
Combine libraries with Speed-Beads at a 1:1 ratio ( 24.5 µL ).
Allow the mixture to incubate at Room temperature for 00:10:00 .
Gently spin plate to collect contents at the bottom of wells.
Place the plate on a plate magnet and wait 00:02:00 -00:03:00 for the solution to clear.
Aspirate the clear supernatant and discard.
Keeping the plate on the magnet, dispense 80% Ethanol into the wells to wash. Ethanol wash volume should be higher than the combined library and Speed-Bead mixture (e.g. 80 µL ).
Incubate 00:00:30 then aspirate to wash.
Repeat for total of 2 washes.
Aspirate final drops of Ethanol with P20 pipette tips. Allow bead pellets to dry on the magnet for 00:01:00 -00:03:00 .
- Dry beads to satin-matte finish, but not to the point of cracking. This happens quickly if ethanol has been aspirated completely.
Remove plate from magnet and dispense Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Allow the mixture to incubate at Room temperature for 00:05:00 .
Gently spin the plate to collect product in the bottom, then place on a plate magnet and wait 00:02:00 -00:03:00 for the eluant to clear.
Remove eluant to a new LoBind plate.
Use a commercial Illumina Library Quantification Kit, following manufacturer's instructions, then pool samples based on calculated molarity.
The library pool is ready to prepare for sequencing.
Option B: Pooling Based on PicoGreen Concentration
1m 30s
Bring Speed-Beads to Room temperature (00:30:00 ).
- Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
Vortex Speed-Beads thoroughly before use.
Combine libraries with Speed-Beads at a 1:1 ratio (24.5 µL ).
Allow the mixture to incubate at Room temperature for 00:10:00 .
Gently spin the plate to collect contents at the bottom of wells.
Place the plate on a plate magnet and wait 00:02:00 -00:03:00 for the solution to clear.
Aspirate the clear supernatant and discard.
Keeping the plate on the magnet, dispense 80% Ethanol into the wells to wash. Ethanol wash volume should be higher than the combined library and Speed-Bead mixture (e.g. 80 µL ).
Incubate 00:00:30 then aspirate to wash.
Repeat for total of 2 washes.
Aspirate final drops of Ethanol with P20 pipette tips. Allow bead pellets to dry on the magnet for 00:00:30 -00:01:00 .
- Dry beads to satin-matte finish, but not to the point of cracking. This happens quickly if ethanol has been aspirated completely.
1m 30s
Remove plate from magnet and dispense 25 µL Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Allow the mixture to incubate at Room temperature for 00:05:00 .
Gently spin the plate to collect product in the bottom of wells, then place on a plate magnet and wait 00:02:00 -00:03:00 for the eluant to clear.
Remove eluant to a new LoBind plate.
Quantify 2 µL of amplified libraries in 48 µL 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.
Add 2 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.
- Add 2 µL of each standard to the appropriate wells.
- Standards 1-8 in duplicate = 16 wells required.
Fill each sample and standard wells with 48 µL 1X PicoGreen Reagent.
- Pipette to mix thoroughly, careful not to introduce bubbles.
Cover plate with optical film and incubate for 00:02:00 at Room temperature before assaying. Protect from light during incubation.
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.
Negative controls can have detectable DNA by PicoGreen in the form of dimers (Step 30.2). To determine the concentration of library in order to pool equimolar volume of each sample, one should use a baseline (averaged NTC concentration) subtracted from the concentration for each sample to determine pooling volumes.
The library pool is ready to prepare for sequencing.
Option C: Pooling Based on Parasitemia
Determine estimated parasitemia of samples by microscopy or real-time PCR.
Bin samples by parasitemia quartile (or other division that makes sense based on parasitemia distribution), recording the number of samples per subpool.
Mix 2uL of each sample for each quartile or subpool into a separate LoBind 1.5mL tube.
You will end up with 4 or more subpools depending on how you group your samples.
Bring Speed-Beads to Room temperature (00:30:00 ).
- Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
Vortex Speed-Beads thoroughly before use.
Combine final pooled libraries with Speed-Beads at a 1:1 ratio in the LoBind 1.5mL tube.
Allow the mixture to incubate at Room temperature for 00:10:00 .
Gently spin tube to collect contents at the bottom.
Place the tube on a tube magnet and wait 00:02:00 -00:03:00 for the solution to clear.
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.
Incubate 00:00:30 then aspirate to wash.
Repeat for total of 2 washes.
Aspirate final drops of Ethanol with P20 pipette tips. Allow bead pellets to dry on the magnet for 00:01:00 -00:03:00 .
- Dry beads to satin-matte finish, but not to the point of cracking.
Remove tube from magnet and dispense Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Allow the mixture to incubate for 00:05:00 .
Gently spin the tube to collect product in the bottom, then place on a tube magnet and wait 00:02:00 -00:03:00 for the solution to clear.
Remove fluid to a new LoBind tube.
Quantify each subpool using a commercial Illumina Library Quantification Kit.
Combine the subpools using the library concentration weighted by the number of samples within each.
Quantify library pool using Qubit dsDNA HS reagent according to the manufacturers protocol.
The library pool is ready to prepare for sequencing.
Option D: Equivolumentric Pooling
Pool 2ul of each library into a LoBind 1.5mL tube. This option does not require SPRI purification prior to pooling libraries.
Bring Speed-Beads to Room temperature (00:30:00 ).
- Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) and molecular grade water.
Vortex Speed-Beads thoroughly before use.
In a LoBind 1.5mL tube, combine final pooled libraries with Speed-Beads at a 1:1 ratio.
Allow the mixture to incubate at Room temperature for 00:10:00 .
Gently spin tube to collect contents at the bottom.
Place the tube on a tube magnet and wait 00:02:00 -00:03:00 for the solution to clear.
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.
Incubate 00:00:30 then aspirate to wash.
Repeat for total of 2 washes.
Aspirate final drops of Ethanol with P20 pipette tips. Allow bead pellets to dry on the magnet for 00:01:00 -00:03:00 .
- Dry beads to satin-matte finish, but not to the point of cracking.
Remove tube from magnet and dispense Elution Buffer onto each bead pellet. Mix by pipetting to resuspend DNA.
Allow the mixture to incubate for 00:05:00 .
Gently spin the tube to collect product in the bottom, then place on a tube magnet and wait 00:02:00 -00:03:00 for the solution to clear.
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.