May 19, 2026
Version 2
IDEEL- UNC Implemented 4CAST Modification (Nextera Library Preparation Protocol) V.2
- 1University of North Carolina at Chapel Hill
- IDEEL
Protocol Citation: Jonathan Juliano, Jacob Sadler 2026. IDEEL- UNC Implemented 4CAST Modification (Nextera Library Preparation Protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4qdmlx9/v2Version created by Jacob Sadler
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: April 14, 2026
Last Modified: May 19, 2026
Protocol Integer ID: 315032
Keywords: Infectious Disease Epidemiology and Ecology Lab, IDEEL, 4CAST amplicon panel, Multiplexed amplicon sequencing, Nextera Library Preparation, nextera library preparation protocol, 4cast modification of nextera library preparation, nextera library preparation, 4cast modification, protocol detail, protocol, Plasmodium falciparum, P. falciparum, falciparum, Plasmodium, CSP, AMA1, protocol
Abstract
This protocol details the 4CAST modification of Nextera Library Preparation.
Guidelines
Overview:
This protocol is designed for use with the current 4CAST amplicon panel. The original publication of this protocol is: LaVerriere, E., Schwabl, P., Carrasquilla, M., Taylor, A. R., Johnson, Z. M., Shieh, M., et al. (2022). Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study. Mol Ecol Resour 22, 2285–2303.
Portions of this protocol are taken verbatim from Supplementary Protocol S1 from this manuscript.
PCR assays are “Nextera-Adapted” by adding standardized, identical 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
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.
4CAST Primers
Primer To Order
4-CSP-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTAAGGAACAAGAAGGATAATACCA
4-CSP-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAATGACCCAAACCGAAATG
4-TRAP-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCAGCACATGCGAGTAAAG
4-TRAP-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACCCGAAAATAAGCACGA
4-SERA-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTACTTTCCCTTGCCCTTGTG
4-SERA-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACTACAGATGAATCTGCTACAGGA
4-AMA-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCATCAGGGAAATGTCCAGT
4-AMA-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCCTGCATGTCTTGAACA
I7 Index primers
| 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 |
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 |
Fragment Analysis of Final Library from Field Isolates - Agilent D1000 High Sensitivity Tape Station
Materials
Consumables:
1. P1000 pipettor and tips
2. P200 pipettor and tips; single and multichannel
3. P20 pipettor and tips ; single and multichannel
4. 25µL reagent reservoir (3)
5. 8-tube strips
6. 96-well PCR plates
7. 1.5mL Eppendorf tubes
8. 50mL conical tubes
Reagents:
1. Anhydrous Ethanol
2. Molecular Grade Water (MGW)
3. SPRI Beads
4. KAPA HiFi HotStart ReadyMix 2X
5. Pre-Combined UDI 96-well plate @ 10µM each index (i7 and i5 Index primers)
6. 4CAST Primers (see Guidelines & Warnings section for primer
Before Beginning
Reconstitute all primers to 100 µM per manufacturer instructions.
Prepare the thermocycler by programming the following conditions for PCR1 and Library Prep PCR.
PCR1:
Heat lid to 105 °C .
Library Prep PCR:
Heat lid to 105 °C .
4CAST Primer Mixture Preparation
Combine 10 µL of each 100 micromolar (µM) primer (forward and reverse for CSP, AMA1, SERA2, and TRAP) in a 1.5mL eppendorf tube.
Note
Primer mixture can be stored at -20 °C .
4CAST Primer Mixture Dilution
In a clean tube prepare a 1:1 dilution of primer mixture to MGW (molecular grade water) achieving a working concentration of 6.250 micromolar (µM) of each primer.
PCR Reaction 1
Calculate the amount of reagents needed for the number of samples to be run plus 3 additional reactions for controls. Use a 10% excess to account for pipette loss.
| A | B | |
| Reagent | Volume per Reaction (µL) | |
| KAPA HiFi HotStart ReadyMix 2X | 5.0 µL | |
| Primer Cocktail | 1.5 µL | |
| Total MM Volume | 6.5 µL |
Add 4.0 µL sample genomic DNA (or negative/positive controls) to each well. Gently mix by pipetting up and down 3X
Place the plate on the thermocycler and run the following program:
Heat lid to 105 °C .
PCR1 Clean-up
Equilibrate SPRI beads to room temperature.
Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) molecular grade water. Each sample will need a total 80% Ethanol volume of 100µL.
Spin down the PCR1 plate to ensure all liquid is at the bottom of the wells.
Add 10 µL SPRI beads to each reaction. No need to mix. Cover thoroughly with plate seal and vortex gently. Incubate atRoom temperature for 10 minutes.
After incubation spin plate thoroughly so all liquid is at the bottom of the wells.
Place plate on a 96-well magnet and allow beads to capture for 2 minutes. Aspirate clear supernatant and discard.
Dispense 50 µL of 80% Ethanol into each well. Incubate at Room temperature for 30 seconds.
Aspirate and dispose of ethanol wash.
Repeat step 13 for a total of 2 washes.
Aspirate final drops of ethanol with P20 pipette tips
Resuspend beads using 10 µL MGW. Incubate for 5 minutes at Room temperature to elute 4CAST amplicons.
Transfer purified amplicons to a clean plate.
Note
Purified amplicons can be stored short-term at -20 °C prior to Library Preparation PCR.
Setup for Library Preparation PCR: Preparation of Materials
Generation of UDI plates is described in Step 19 at dx.doi.org/10.17504/protocols.io.e6nvwbwy7vmk/v2.
Thaw Pre-Combined UDI 96-well plate, purified PCR reaction, and KAPA Master Mix.
Record the combinatorial index plate used (e.g. UDI-A1, UDI-C3, UDI-E5).
Spin the UDI and purified PCR 1 plates thoroughly prior to opening.
Library Preparation PCR
Prepare a master mix using the following reaction volumes with 10% excess. Distribute 7.0 µL of master mix into each well.
| A | B | |
| Reagent | Volume per Reaction (µL) | |
| KAPA HiFi HotStart ReadyMix 2X | 5.0 µL | |
| Molecular H2O | 2.0 µL | |
| Total Volume | 7.0 µL |
Divide total volume by 8 or 12 into strip tubes then use multichannel to distribute into the PCR plate
Add 2.2 µL of the appropriate 10 micromolar (µM) UDI.
Add 3.0 µL of PCR 1 product to each well then seal plate thoroughly.
Place the plate in the thermocycler and run the following program:
Heat lid to 105 °C .
Library
Note
Library can be frozen at -20 °C at this point.
Sample pooling and double-sided SPRI bead clean-up/size selection
20m
Equilibrate SPRI beads to Room temperature for 00:30:00 prior to use; take Tapestation reagents out of 4℃ to come to room temperature prior to use (if using).
Combine approximately 8 µL of each sample into a 2.0mL Eppendorf tube to create the sample pool. For high-throughput applications you may decrease pooling volume to 3µL
If completing with less than 13 samples, can adjust volume of each sample accordingly (ex: use 10 µL of each sample) to achieve an adequate sample pool volume.
Mix gently and spin down.
Perform bead clean-up using SPRI beads for size selection.
Transfer 100 µL combined sample into a PCR tube and add 55 µL beads, mixing well.
Incubate at Room temperature for 00:10:00 .
5m
Place on a magnetic rack for 00:03:00 .
5m
KEEP supernatant. Transfer supernatant into a new PCR tube.
Add 20 µL of beads to the transferred supernatant, mixing well. Incubate at Room temperature for 00:05:00 .
5m
Place on the magnetic rack for 00:03:00 .
3m
Keeping the tube on the magnetic rack, discard supernatant and wash twice with 80% ethanol.
- Use approximately 150 µL for each wash.
Elute in 30 µL 10 millimolar (mM) Tris-HCl pH 8.0. Incubate for 00:03:00 , place on the magnetic rack, and allow the pellet to form (about a minute).
- If using stock Tris-HCl, the concentration is often 1M; therefore, dilute down to 10 millimolar (mM) with 1 µL of Tris-HCl into 99 µL molecular grade water.
2m
Transfer eluate and to a clean 1.5mL eppendorf tube.
Add 2.5 µL 10.0 millimolar (mM) Tris-HCl containing 0.05% Tween-20 for final suspension.
QC/library selection for sequencing
A full qPCR quantification is not necessary for 4CAST.
We recommend performing Qubit HS dsDNA quantitation and a D1000 Agilent tape station on the final library to confirm fragment size. See expected fragment analysis within the Guidelines & Warnings section.