Oct 16, 2025
IDEEL- UNC Implemented 4CAST Modification (Nextera Library Preparation Protocol)
- Jonathan Juliano1
- 1University of North Carolina
- IDEEL
Protocol Citation: Jonathan Juliano 2025. IDEEL- UNC Implemented 4CAST Modification (Nextera Library Preparation Protocol). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4qdmlx9/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 13, 2025
Last Modified: October 16, 2025
Protocol Integer ID: 229871
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
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 |
Materials
Consumables:
1. P200 pipettor and tips
2. P20 pipettor and tips
3. 25µL reagent reservoir (3)
4. 96-well PCR plates
Reagents:
1. 80% Ethanol
2. AmpureXP beads
3. KAPA HiFi HotStart ReadyMix 2X
4. Molecular Grade Water (MGW)
5. Pre-Combined UDI 96-well plate @ 10µM each index (i7 and i5 Index primers)
6. 4CAST primer
Troubleshooting
4CAST Primer Mix Preparation
Combine 10 µL of each primer (forward and reverse for CSP, AMA1, SERA2, and TRAP) at 100 micromolar (µM) into 1.5mL Eppendorf tube.
Store primer cocktail at -20 °C .
Volume will need to be modified if more than 96 samples are being prepared at once.
Perform a 1:1 dilution with MGW to achieve a working concentration of 6.250 micromolar (µM) of each primer.
For primer mix with 10 µL of each primer, use 80 µL of primer mix and 80 µL dH2O.
PCR1
Calculate the amount of reagents needed with a 10% excess to account for pipette loss.
Prepare the PCR1 cocktail using the following quantities for each sample being prepared and pipet 6.5 µL of mix into each well of a 96-well PCR plate.
| A | B | |
| REAGENT | VOLUME/REACTION | |
| 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 templates) to each well.
Recommended to place controls randomly throughout the plate.
Place the plate on a thermocycler and program with the following conditions:
Setup for Library Preparation PCR: Preparation of Materials
Generation of UDI plates is described at 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.
Mix fresh 80% Ethanol using 200 proof ethanol (100%, anhydrous) molecular grade water.
Record the combinatorial index plate used (e.g. UDI-A1, UDI-C3, UDI-E5).
Setup for Library Preparation PCR: Preparing UDI Plate
Spin UDI plate to ensure there is no liquid on the plate sealing foil.
Remove foil from desired UDI wells row-by-row or column-by-column, using a razor to carefully score between wells.
Use the edge of the razor to lift the foil before gently peeling the foil off.
This method allows for easy tracking of what indexes have been added and helps limit contamination of index plates.
Dilute UDI in plate as necessary to reach a concentration of 10 micromolar (µM) .
Add 40 µL Tris with no EDTA (if possible) for dilution from UDI plates.
Perform dilution in the UDI plate.
Add 2.2 µL of the appropriate UDI to each well. No need to mix yet.
Record which well of the UDI plate was used for each sample.
Seal UDI plate with a fresh piece of foil immediately after use.
Library Preparation PCR
Calculate the amount of master mix reagents needed with a 10% excess to account for pipette loss. (Use tool here for calculations)
Prepare LP-PCR plate for indexing samples with the following reaction volumes.
| A | B | |
| REAGENT | VOLUME/REACTION | |
| KAPA HiFi HotStart ReadyMix 2X | 5.0 µL | |
| Molecular H2O | 2.0 µL | |
| Unique Dual Index | 2.2 µL | |
| Total MM Volume | 9.2 µL |
Add 3.0 µL of PCR 1 product to each well.
| A | B | C | D | |
| STEP | # CYCLES | TEMP C | TIME | |
| Activation | 1 | 95 | 1 min. | |
| -Denaturation | 15 | 95 | 15 sec. | |
| -Annealing | 55 | 15 sec | ||
| -Extension | 72 | 30 sec | ||
| Final Extension | 1 | 72 | 1 min. | |
| hold | 1 | 4 | forever |
Place the plate on a thermocycler and program with the following conditions:
Note
Sample can be frozen at -20 °C at this point.
Sample pooling and double-sided clean-up/size selection
20m
Note
Equilibrate 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.
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 AmpureXP 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 00:05:00 .
5m
Place on a magnetic rack for 00:05:00 .
5m
KEEP and remove supernatant from beads and transfer into a new PCR tube.
Add 20 µL of beads, mixing well and incubating at Room temperature for 00:05:00 .
5m
Place on the magnetic rack for 00:03:00 .
3m
Discard supernatant and wash twice with 80% ethanol while still on rack.
- Use approximately 150 µL for each wash.
Elute in 30 µL 10 millimolar (mM) Tris-HCl pH 8.0, incubate for 00:02:00 , place on the magnetic rack, and allow the pellet to form (about 3 minutes).
- If using stock Tris-HCl, concentration is typically 1M; therefore, dilute down to 10 millimolar (mM) with 1 µL of Tris-HCl into 99 µL molecular grade water.
2m
Keep and remove supernatant and transfer to a 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:
Qubit
Tapestation
QC can be done by the core or sequencing facility.