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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 protocol</span><span style = "vertical-align:super;">1</span><span>. The library preparation is Illumina NexteraXT. Library pooling and normalization were adapted from the Gohl protocol</span><span style = "vertical-align:super;">3</span><span>. </span></div><div class = "text-block">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.</div></div>

[STEPS]
?. [RNA Extraction]
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.
?. [cDNA Generation]
cDNA from RNA from any extraction method is produced using   with the following ratios, per sample:
[SuperScript IV VILO Master Mix ]
[Nuclease-free water]
[Viral RNA]
?. [cDNA Generation]
Tightly seal reaction wells, mix reaction components with plate mixer and spin down
?. [cDNA Generation]
Run the following thermal cycler protocol:
[10 minutes]
[10 minutes]
[5 minutes]
[∞]
?. [ARTIC Amplicon Generation]
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:
[Q5 HI-FIDEL 2X MASTER MIX 500 rxn				]
[IDT ARTIC V3 Primer Pool (20uM)]
[nuclease-free water]
[cDNA]
?. [cDNA Generation]
Store cDNA at -20ºC
?. [ARTIC Amplicon Generation]
Tightly seal reaction wells, mix reaction components with plate mixer and spin down
?. [ARTIC Amplicon Generation]
Run the following thermal cycler protocol:     total of 35 cycles of steps 2 and 3
[30 seconds]
[15 seconds]
[5 minutes]
[∞]
?. [ARTIC Amplicon Generation]
Combine PCR-amplified DNA from primer pool 1 and 2 together and dilute to 0.2-0.6ng/µL (for Illumina)
?. [ARTIC Amplicon Generation]
Proceed to Illumina library prep method of choice (NexteraXT, Flex, Illumina compatible)
?. [Library Quantification & Pooling]
Quantify the DNA concentration of each clean library using the Qubit High Sensitivity dsDNA kit
?. [Library Quantification & Pooling]
Pool equal concentrations (e.g., 1-10 ng) of each library. Total volume does not matter
?. [Library Quantification & Pooling]
Concentrate using 0.7xAMPureXPbeads (ex. for 240 µL add 168 µL of beads)
?. [Library Quantification & Pooling]
Allow binding at room temperature for at least 5 minutes before clearing with magnet
?. [Library Quantification & Pooling]
Wash beads 2x with 80% EtOH while still on magnet
?. [Library Quantification & Pooling]
Remove all EtOH and allow to pellet to dry for 5 minutes
?. [Library Quantification & Pooling]
Remove tube from magnet and add 20 µL Tris-HCl pH 8.0 to pellet. Slowly pipette mix
?. [Library Quantification & Pooling]
Incubate at room temperature for at least 5 minutes before clearing with magnet
?. [Library Quantification & Pooling]
Check DNA fragment distributions of the pooled sample. Peak fragment size from 400 bp tiled amplicons with proper ligated adaptors should be ~ 500nt
?. [Library Quantification & Pooling]
Quantify the DNA concentration of the pooled library using the Qubit High Sensitivity DNA kitNote: At least 0.76 ng/µL is required to achieve 2nM for library pooling. Libraries will need to be concentrated or re-amplified if less than this amount.
?. [Library Quantification & Pooling]
Convert DNA libraries from weight to moles:Molecular weight [nM] = Library 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 = 0.3822.5 / 0.382= 6.5nM
?. [Library Quantification & Pooling]
Dilute the pooled library to 2nM in 10 mM Tris pH 8.0
?. [Final Dilution and Loading]
Add 10µLof a 2nM library to 10 µL of freshly prepared 0.2 N NaOH
?. [Final Dilution and Loading]
Mix by flicking, spin down, incubate at room temperature for 5 minutes
?. [Final Dilution and Loading]
Add 980µl prechilled HT1to the tube containing denatured library. The result is 1 ml of a 20pM denatured library
?. [Final Dilution and Loading]
Add 240 µL of the 20pM denatured library, 348 µl prechilled HT1, and 12 µL of 20pM denatured PhiX. Invert to mix, spin down
?. [Final Dilution and Loading]
The result is 600 µL of an 8pM denatured library with 5% PhiX ready to be loaded on a MiSeq v3 cartridge
?. [References]
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.bez7jf9n2. 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/s13059-018-1618-73. Daryl Gohl2020.SARS-CoV-2 Tailed Amplicon Illumina Sequencing.protocols.iohttps://dx.doi.org/10.17504/protocols.io.bipikdke4. 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