BPHL SARS-CoV-2 Tiled Amplicon Illumina Sequencing

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 from RNA (from any extraction method) is produced using with the following ratios, per sample:

4µL

6µL

10µL

Tightly seal reaction wells, mix reaction components with plate mixer and spin down

Run the following thermal cycler protocol:

25°C

50°C

85°C

4°C

Store cDNA at -20ºC

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:

12.5µL

1µL

9µL

2.5µL

Tightly seal reaction wells, mix reaction components with plate mixer and spin down

Run the following thermal cycler protocol:

98°C

95°C

64°C

 total of 35 cycles of steps 2 and 3

4°C

Combine PCR-amplified DNA from primer pool 1 and 2 together and dilute to 0.2-0.6 ng/µL (for Illumina)

Proceed to Illumina library prep method of choice (NexteraXT, Flex, Illumina compatible)

Quantify the DNA concentration of each clean library using the Qubit High Sensitivity dsDNA kit

Pool equal concentrations (e.g., 1-10 ng) of each library. Total volume does not matter

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

Dilute the pooled library to 2 nM in 10 mM Tris pH 8.0

Concentrate using 0.7x AMPureXP beads (ex. for 240 µL add 168 µL of beads)

Allow binding at room temperature for at least 5 minutes before clearing with magnet

Wash beads 2x with 80% EtOH while still on magnet

Remove all EtOH and allow to pellet to dry for 5 minutes

Remove tube from magnet and add 20 µL Tris-HCl pH 8.0 to pellet. Slowly pipette mix

Incubate at room temperature for at least 5 minutes before clearing with magnet

Check DNA fragment distributions of the pooled sample. Peak fragment size from 400 bp tiled amplicons with proper ligated adaptors should be ~ 500nt

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.

Add 10 µL of a 2 nM library to 10 µL of freshly prepared 0.2 N NaOH

Mix by flicking, spin down, incubate at room temperature for 5 minutes

Add 980 µl prechilled HT1to the tube containing denatured library. The result is 1 ml of a 20 pM denatured library 

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

The result is 600 µL of an 8 pM denatured library with 5% PhiX ready to be loaded on a MiSeq v3 cartridge

1. Nate Matteson, Nathan Grubaugh, Karthik Gangavarapu, Josh Quick, Nick Loman, Kristian Andersen2020.PrimalSeq: Generation of tiled virus amplicons for MiSeq sequencing. protocols.io https://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.io https://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