TITLE: SARS-CoV-2 Tailed Amplicon Illumina Sequencing
AUTHORS: Daryl Gohl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol outlines how to process RNA for SARS-CoV-2 sequencing using tailed primers to generate tiled amplicons using the method described here: </span><a href="https://www.biorxiv.org/content/10.1101/2020.05.11.088724v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2020.05.11.088724v1</span></a><span style = ":;">.</span></div><div class = "text-block"><span>Best results are obtained for samples with N1 and N2 Ct values of <30 (based on the UMGC/MDL implementation of the CDC qRT-PCR diagnostic assay for SARS-CoV-2, see here: </span><a href="https://www.biorxiv.org/content/10.1101/2020.04.02.022186v1.full" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2020.04.02.022186v1.full</span></a><span>). For samples with N1 and N2 values between 30 and ~35, coverage and other sequencing metrics may be more variable and increased adapter dimer formation is expected.</span></div></div>

[STEPS]
?. [Set up]
Clean workspace and pipets by spraying with RNaseZAP or comparable product (such as RNase Away) and wiping down with KimWipes prior to beginning work.RNA samples should be stored at  and thawed on ice.
-80 °C
?. [cDNA synthesis]
Thaw RNA samples on ice then transfer of sample into a 96-well Thermo PCR plate.
5 µl
?. [cDNA synthesis]
Set up the following reverse transcription reaction master mix (multiply below volumes by number of reactions plus desired overage):
[nuclease free water]
[SuperScript IV VILO master mix]
?. [cDNA synthesis]
Transfer  of reverse transcription master mix to each sample containing well.
15 µl
?. [cDNA synthesis]
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for s, and spin down briefly in a plate centrifuge (s at.
Centrifuge: 2500 33
?. [cDNA synthesis]
Incubate in a thermocycler using the following conditions: for  for  for
25 °C
50 °C
85 °C
?. [Enrichment PCR]
Transfer  of cDNA to each of 4 96-well Thermo PCR plates labeled: Project_Name_PCR1_1.1, Project_Name_PCR1_1.2, Project_Name_PCR1_2.1, and Project_Name_PCR1_2.2.
2.5 µl
?. [Enrichment PCR]
Set up the following four PCR master mixes, one for each of the four multiplexed primer pools (multiply below volumes by number of reactions plus desired overage):  (Either pool 1.1, 1.2, 2.1, or 2.2)
[ nuclease-free water]
[5x Q5 reaction buffer ]
[10mM dNTPs]
[Q5 Polymerase ]
[primer pool (10 µM)]
?. [Enrichment PCR]
Transfer  of master mix to each well of the appropriate PCR plate.
22.5 µl
?. [Enrichment PCR]
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for , and spin down briefly in a plate centrifuge ( at ).
Centrifuge: 2500 33
?. [Enrichment PCR]
Amplify samples using the following PCR conditions: for 	35 cycles of: for  for
98 °C
98 °C
65 °C
?. [Indexing PCR]
For each sample, combine  of each of the four pools in a single Bio-Rad fully-skirted 96 well plate.
10 µl
?. [Indexing PCR]
Seal plate with a “F” seal, mix well by vortexing using a plate vortexer at 1900 rpm for , and spin down in a plate centrifuge ( at ).
Centrifuge: 2500 33
?. [Indexing PCR]
In a 96-well Thermo plate, add  of each sample to  of nuclease free water (1:100 dilution).
2 µl
198 µl
?. [Indexing PCR]
Seal plate with a “F” seal, mix well by vortexing using a plate vortexer at 2500 rpm for , and spin down in a plate centrifuge (  at ).
Centrifuge: 2500 33
?. [Indexing PCR]
Transfer  of 1:100 diluted PCR 1 sample to a 96-well Thermo PCR plate.
5 µl
?. [Indexing PCR]
Transfer  of 5 µM indexing primer mix to the 96-well Thermo PCR plate containing the samples.
2 µl
?. [Indexing PCR]
Set up the following PCR master mix (multiply below volumes by number of reactions plus desired overage):
[ nuclease-free water]
[5x Q5 reaction buffer ]
[10 mM dNTPs ]
[Q5 Polymerase]
?. [Indexing PCR]
Transfer  of master mix to each well of the appropriate PCR plate.
3 µl
?. [Indexing PCR]
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for , and spin down briefly in a plate centrifuge ( at ).
Centrifuge: 2500 33
?. [Indexing PCR]
Amplify samples using the following PCR conditions: for 	10 cycles of: for  for for  for
98 °C
98 °C
55 °C
72 °C
72 °C
?. [Normalization]
Normalize samples using a SequalPrep plate according to manufacturer’s instructions.
?. [Normalization]
Elute in  of SequalPrep Elution Buffer.
20 µl
?. [Pooling]
Pool  of each sample in a trough, mix well and transfer material to a  non-stick tube.
10 µl
1.5 mL
?. [Pooling]
Purify using AMPureXP beads at a 0.7x ratio. Elute library in  of EB.
20 µl
?. [Library QC]
Perform final QC on pool by determining concentration (PicoGreen or Qubit assay). Prepare 2 nM pool dilution, based on the sample concentration as determined by PicoGreen and fragment size (expected size is ~555 bp).
?. [Sequencing]
Dilute pooled sample to 8 pM in HT1, following MiSeq loading instructions, spike in 5% 8 pM PhiX, and load in MiSeq 2x250 or 2x300 reagent cartridge.