TITLE: BPHL SARS-CoV-2 Tiled Amplicon Illumina Sequencing 
AUTHORS: Jason  Blanton

[DESCRIPTION]
This protocol details the Florida Department of Health's Bureau of Public Health Laboratories' (BPHL) wet lab portion of our SARS-CoV-2 next generation sequencing workflow. The method is a tiled amplicon approach using ARTIC V3 primers. The amplicon generation was adapted from the Matteson protocol1. The library preparation is Illumina NexteraXT. Library pooling and normalization were adapted from the Gohl protocol3. 

This protocol is for loading a MiSeq, but we have had equal success running on iSeqs and NextSeqs as well. Up to 96 libraries can be run on a MiSeq and up to 384 on a NextSeq.


[STEPS]
SECTION: RNA Extraction
1. Extract RNA from positive COVID-19 clinical specimens with the KingFisher Flex instrument using the Applied Biosystems™MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit and its associated protocol.
SECTION: cDNA Generation
2. cDNA from RNA (from any extraction method) is produced using    with the following ratios, per sample:

4 µL 
6 µL 
10 µL 


SECTION: cDNA Generation
2.1. Tightly seal reaction wells, mix reaction components with plate mixer and spin down
SECTION: cDNA Generation
2.2. Run the following thermal cycler protocol:

25 °C 
50 °C 
85 °C
4 °C 

SECTION: cDNA Generation
2.3. Store cDNA at -20ºC 
SECTION: ARTIC Amplicon Generation
3. ARTIC amplicons are produced by preparing two PCR reactions per sample (primer pool 1 is one reaction, primer pool 2 is the other). Per sample, combine below reagents in the listed ratios:

12.5 µL 
1 µL 
9 µL 
2.5 µL 

SECTION: ARTIC Amplicon Generation
3.1. Tightly seal reaction wells, mix reaction components with plate mixer and spin down
SECTION: ARTIC Amplicon Generation
3.2. Run the following thermal cycler protocol:
98 °C 
95 °C 
64 °C 
     total of 35 cycles of steps 2 and 3
4 °C 

SECTION: ARTIC Amplicon Generation
3.3. Combine PCR-amplified DNA from primer pool 1 and 2 together and dilute to 0.2-0.6 ng/µL (for Illumina)
SECTION: ARTIC Amplicon Generation
3.4. Proceed to Illumina library prep method of choice (NexteraXT, Flex, Illumina compatible)
SECTION: Library Quantification &amp; Pooling
4. Quantify the DNA concentration of each clean library using the Qubit High Sensitivity dsDNA kit
SECTION: Library Quantification &amp; Pooling
4.1. Pool equal concentrations (e.g., 1-10 ng) of each library. Total volume does not matter
SECTION: Library Quantification &amp; Pooling
4.2. Concentrate using 0.7x AMPureXP beads (ex. for 240 µL add 168 µL of beads)
SECTION: Library Quantification &amp; Pooling
4.3. Allow binding at room temperature for at least 5 minutes before clearing with magnet
SECTION: Library Quantification &amp; Pooling
4.4. Wash beads 2x with 80% EtOH while still on magnet
SECTION: Library Quantification &amp; Pooling
4.5. Remove all EtOH and allow to pellet to dry for 5 minutes
SECTION: Library Quantification &amp; Pooling
4.6. Remove tube from magnet and add 20 µL Tris-HCl pH 8.0 to pellet. Slowly pipette mix
SECTION: Library Quantification &amp; Pooling
4.7. Incubate at room temperature for at least 5 minutes before clearing with magnet
SECTION: Library Quantification &amp; Pooling
4.8. Check DNA fragment distributions of the pooled sample. Peak fragment size from 400 bp tiled amplicons with proper ligated adaptors should be ~ 500nt
SECTION: Library Quantification &amp; Pooling
4.9. Quantify the DNA concentration of the pooled library using the Qubit High Sensitivity DNA kit

Note: At least 0.76 ng/µL is required to achieve 2 nM for library pooling. Libraries will need to be concentrated or re-amplified if less than this amount.
SECTION: Library Quantification &amp; Pooling
4.10. Convert DNA libraries from ng/uL to moles:
concentration [nM] = concentration [ng/µL] / ((ave.library size x 660)/1,000,000)

Example: if average size of library is 580 bp and concentration is 2.5 ng/µL:
(580 x 660) / 1,000,000 = 0.382
2.5 / 0.382 = 6.5 nM
SECTION: Library Quantification &amp; Pooling
4.11. Dilute the pooled library to 2 nM in 10 mM Tris pH 8.0
SECTION: Final Dilution and Loading
5. Add 10 µL of a 2 nM library to 10 µL of freshly prepared 0.2 N NaOH
SECTION: Final Dilution and Loading
5.1. Mix by flicking, spin down, incubate at room temperature for 5 minutes
SECTION: Final Dilution and Loading
5.2. Add 980 µl prechilled HT1to the tube containing denatured library. The result is 1 ml of a 20 pM denatured library 
SECTION: Final Dilution and Loading
5.3. Add 240 µL of the 20 pM denatured library, 348 µl prechilled HT1, and 12 µL of 20 pM denatured PhiX. Invert to mix, spin down
SECTION: Final Dilution and Loading
5.4. The result is 600 µL of an 8 pM denatured library with 5% PhiX ready to be loaded on a MiSeq v3 cartridge

SECTION: References
6. 1. Nate Matteson, Nathan Grubaugh, Karthik Gangavarapu, Josh Quick, Nick Loman, Kristian Andersen2020.PrimalSeq: Generation of tiled virus amplicons for MiSeq sequencing.protocols.iohttps://dx.doi.org/10.17504/protocols.io.bez7jf9n

2. Grubaugh, ND. et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biology  20,8 (2019) https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7

3. Daryl Gohl2020.SARS-CoV-2 Tailed Amplicon Illumina Sequencing.protocols.iohttps://dx.doi.org/10.17504/protocols.io.bipikdke

4. Gohl DM,  Garbe J,  Grady P,  Daniel J,  Watson RHB,  Auch B,  Nelson A,  Yohe S,  Beckman KB, A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2. BMC Genomics  doi: 10.1186/s12864-020-07283-6