Oct 30, 2025
IDEEL- UNC Implementation of DBLa Var-Typing
- Infectious Disease Epidemiology and Ecology Lab1
- 1Infectious Disease Epidemiology and Ecology Lab, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- IDEEL
Protocol Citation: Infectious Disease Epidemiology and Ecology Lab 2025. IDEEL- UNC Implementation of DBLa Var-Typing. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n781lwz/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: October 27, 2025
Last Modified: October 30, 2025
Protocol Integer ID: 231055
Keywords: DBLa PCR, Amplification, Sequencing, Primers, unc implementation of dbla var, dbla var, continued plasmodium falciparum disease transmission, pcr assay, plasmodium falciparum disease transmission after an outbreak, use with the current dbla, molecular epidemiology, existing primer sequence, current dbla, pcr, primer sequence, sequencing approach, adapted primer, reverse primer, forward primer, standardized oligo segment, example publication of this protocol
Abstract
This protocol is designed for use with the current DBLa sequencing approach developed by the Day laboratory. An example publication of this protocol is: Ruybal-Pesántez S, Sáenz FE, Deed SL, Johnson EK, Larremore DB, Vera-Arias CA, Tiedje KE, Day KP. Molecular epidemiology of continued Plasmodium falciparum disease transmission after an outbreak in Ecuador. Front Trop Dis. 2023;4:1085862. doi: 10.3389/fitd.2023.1085862. Epub 2023 Mar 16. PMID: 39525803; PMCID: PMC11546077.
PCR assays are “Nextera-Adapted” by adding standardized oligo segments onto existing primer sequences.
For example:
- Non-Nextera-Adapted Primer:
- Forward primer: CCATCAGGGAAATGTCCAGT
- Reverse primer : TTTCCTGCATGTCTTGAACA
- Forward primer addition: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
- Reverse primer addition: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
Example:
- Nextera-Adapted Primer:
- Forward: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCATCAGGGAAATGTCCAGT
- Reverse: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCCTGCATGTCTTGAACA
PCR-based library preparation allows for a significant reduction in time to complete libraries, reduces the consumables required, and lessens the impact of library preparation on technicians. Pre-combined 96-well Unique Dual Index plates are utilized in combination with standardized barcoding worksheets to simplify the generation of final library indexing manifests.
Materials
Materials
Consumables:
- P200 pipettor and tips/ single or multichannel
- P20 pipettor and tips/ single or multichannel
- 25mL reagent reservoir
- 96-well PCR plates
- Nuclease-free PCR microtubes/strips/plates.
- Nuclease-free 1.5µL Eppendorf tubes (for master mixes).
- Cold blocks for microplates and 1.5µL tubes.
Reagents:
- 80% Ethanol
- Qubit or PicoGreen supplies for quantifying product
- Roche Fast Start High Fidelity PCR System (Cat #: 03553400001)
- Molecular Grade Water (MGW)
- Pre-Combined UDI 96-well plate at 10µM each index (i7 and i5 Index primers)
- DBLa primers
- KAPA HiFi HotStart ReadyMix 2X (Cat #: 07958927001)
Materials: DBLa Primers
| A | B | |
| Primer | Sequence | |
| Nextera DBLaF | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCACGMAGTTTYGC | |
| NexteraDBLaR | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCCATTCSTCGAACCA |
Materials: I7 Index primers
| 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 |
Materials: I5 Index Primers
| 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 |
Troubleshooting
Step 1: General Lab measures
Ensure all work areas and pipettors are sterilized per laboratory protocol.
Note
- Clean pipettes and working bench tops with 70% ethanol.
- UV-treat the working surface, all equipment, and tubes/strips.
DBLa PCR (PCR1) should be set up in a clean room, free of PCR amplification.
Indexing PCR (PCR2), clean-up, and quantification should be done in an area where PCR products can be worked with (i.e., a post-PCR space).
Thaw reagents and DNA samples On ice .
Briefly vortex and centrifuge each reagent before use, with the exception of enzymes.
Note
Gently mix enzymes by inverting the tube a few times, then centrifuge.
Step 2: Setting up Primers
Dilute the 100 micromolar (µM) stock primers down to 20 micromolar (µM) working solutions for DBLa forward and reverse primers.
Generation of UDI Plates
The final concentration of the i5/i7 primer mix should be 10 micromolar (µM) . This can be done any number of ways, including diluting the 100 micromolar (µM) stock of each primer to 10 micromolar (µM) and then mixing.
UDI Combinatorial Plates can be made ahead of time and stored at -20 °C for use.
Typically, we recommend making plates for a maximum of 10 indexing PCR reactions; thus, if contamination should occur, a new plate can be made without wasting reagents.
Figure legend: 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.
Step 3: Initial DBLa PCR
Calculate the amount of reagents needed with a 10% excess to account for pipette loss.
Prepare the PCR master mix based on the table below.
| A | B | |
| Reagent | Volume/rxn (µL) | |
| Roche Fast Start High Fidelity PCR System Buffer (2X) | 2.5 | |
| Nucleotide Mix | 0.5 | |
| Fwd Primer (20uM) | 2.5 | |
| Rev Primer (20uM) | 2.5 | |
| Enzyme Blend | 0.2 | |
| Water | 14.8 |
Note
- All reagents, PCR master mix, primer pools, and samples should be held in freezer blocks (tube or 96-well) unless stated otherwise.
- 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 the samples to be tested. For non-template control, we recommend using human DNA at a clinically relevant concentration (e.g. 16.77 ng/µL). If human DNA is not available, water can be used as a non-template control.
Combine the master mix and 2 µL sample DNA (25µl total reaction volume), seal caps/strips/plates, and place the reaction in a thermal cycler using the following conditions:
| A | B | C | D | |
| Step | Duration | Cycles | Temperature | |
| Activation | 5 min | 1 cycle | 95° C | |
| 1 min | 1 cycle | 42° C | ||
| 1 min | 1 cycle | 60° C | ||
| Cycling | 1 min | 29 cycles | 95° C | |
| 1 min | 42° C | |||
| 1 min | 60° C |
Step 4: PCR cleanup and library prep
45m
Bring Speedbeads to Room temperature for 00:30:00 .
30m
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 .
Note
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.
10m
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 80% Ethanol with P20 pipette tips. Allow bead pellets to dry momentarily for 00:00:30 to 00:01:30 .
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 eluent and may be added to the library preparation reaction. The eluent is not transferred to a new plate to limit cross-contamination of concentrated amplicon products.
Step 5: Library Preparation (PCR2)
Thaw Pre-Combined UDI 96-well plate, purified PCR reaction, and KAPA Master Mix.
Calculate the amount of reagents needed with a 10% excess to account for pipette loss.
Mix master mix as described in the following table:
| A | B | |
| Reagent | Volume/rxn | |
| KAPA HiFi HotStart ReadyMix 2X | 12.5 µL | |
| Molecular Grade Water | 4.5 µL | |
| Total | 17.0 µL |
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.
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 of PCR1. Gently mix by pipetting.
- Sample and UDI plate should now have the following volumes in each well:
| A | B | |
| Reagent | Volume/rxn | |
| KAPA HiFi HotStart ReadyMix 2X | 12.5 µL | |
| Molecular Grade Water | 4.5 µL | |
| UDI Primer | 2.5 µL | |
| Purified amplicons | 5.0 µL | |
| Total | 24.5 µL |
Program the thermal cycler to 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 |
Step 6: Contamination check
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 possible that negative controls will have a detectable band on gel below the bands consistent with amplification products. Non-template control bands typically represent primer dimer that should not yield data when processed through bioinformatic analysis.
Step 7: Pooling based on PicoGreen Concentration
54m 30s
Bring SpeedBeads to Room temperature ~00:30:00 .
30m
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.
Allow the mixture to incubate at Room temperature for 00:10:00 .
10m
Gently spin the plate to collect contents at the bottom of the wells.
Place the plate on a plate magnet and wait 00:02:00 -00:03:00 for the solution to clear.
3m
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.
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 on the magnet for 00:00:30 to 00:01:00 .
Note
Dry beads to a satin-matte finish, but not to the point of cracking. This happens quickly if ethanol has been aspirated completely.
1m
Remove the plate from the 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 .
5m
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
3m
Remove eluent to a new LoBind plate.
Quantify 2 µL of amplified libraries in 48 µL of 1X PicoGreen reagent in a black, flat bottom microplate appropriate for reading fluorescence.
Note
- If the reagent is not already provided at 1X, combine dye and buffer in a 1:200 ratio 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.
Note
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.
2m
Place the prepared quantitation plate into the multimode plate reader.
Note
The 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 the resulting value by two to report nanograms per microliter.
Negative controls can have detectable DNA by PicoGreen, typically in the form of dimers. To determine the concentration of the library in order to pool equimolar volumes 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.