Jul 17, 2020
Version 2
SARS-CoV-2 Tailed Amplicon Illumina Sequencing V.2
- 1University of Minnesota
- Coronavirus Method Development Community
External link: https://doi.org/10.1186/s12864-020-07283-6
Protocol Citation: Daryl Gohl 2020. SARS-CoV-2 Tailed Amplicon Illumina Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bipikdke
Manuscript citation:
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
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: July 17, 2020
Last Modified: July 17, 2020
Protocol Integer ID: 39370
Abstract
This protocol outlines how to process RNA for SARS-CoV-2 sequencing using tailed primers to generate tiled amplicons using the method described here: https://www.biorxiv.org/content/10.1101/2020.05.11.088724v1.
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: https://www.biorxiv.org/content/10.1101/2020.04.02.022186v1.full). 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.
Materials
1) Fully skirted 96-well plate. (BioRad)
2) Semi-skirted 96-well plate (Thermo Scientific)
3) Nuclease-free water. (Fisher Scientific)
4) Microseal F foil seals. (BioRad)
5) Microseal B PCR seals. (BioRad)
6) SuperScript IV VILO master mix (Thermo)
7) Q5 Hot Start High Fidelity DNA polymerase. (NEB)
8) 10 mM dNTPs (NEB)
9) nCov-2019 pool 1.1, 1.2, 2.1, 2.2 primers. (IDT) – see Appendix
10) Indexing primers. (IDT) – see Appendix
11) Rainin Liquidator 96 pipette with p20/p200 tips. (Rainin)
12) Rainin single/multichannel pipettes with p20/p200/p1000 tips. (Rainin)
13) White Matrix troughs. (Thermo Scientific)
14) SequalPrep Normalization Plate Kit, 96-well. (Thermo Scientific)
15) AMPure XP beads. (Beckman Coulter)
16) Combinatorial Dual Indexing Primers:
For 384 sample barcoding scheme, see “Indexingprimers.xlsx”, from: https://protocolexchange.researchsquare.com/article/nprot-4831/v1
17) Unique Dual Indexing Primers:
Available from Illumina (Nextera Unique Dual Indexing Primers, catalog number: 20027213, 20027214, 20027215, 20027216.
Before start
Tailed primers should be pooled to generate 4 primer pools (1.1, 1.2, 2.1, 2.2) according to the pooling scheme described in Supplemental Data File 2 here:
Set up
Set up
10m
10m
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 -80 °C and thawed on ice.
cDNA synthesis
cDNA synthesis
Thaw RNA samples on ice then transfer5 µL of sample into a 96-well Thermo PCR plate.
Set up the following reverse transcription reaction master mix (multiply below volumes by number of reactions plus desired overage):
11 µL nuclease free water
4 µL SuperScript IV VILO master mix
Transfer 15 µL of reverse transcription master mix to each sample containing well.
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for 00:00:10 s, and spin down briefly in a plate centrifuge (00:00:05 s at2500 rpm .
Incubate in a thermocycler using the following conditions:
25 °C for 00:10:00
50 °C for 00:10:00
85 °C for 00:05:00
Enrichment PCR
Enrichment PCR
Transfer 2.5 µL 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.
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):
14.75 µL nuclease-free water
5 µL 5x Q5 reaction buffer
0.5 µL 10mM dNTPs
0.25 µL Q5 Polymerase
2 µL primer pool (10 µM) (Either pool 1.1, 1.2, 2.1, or 2.2)
Transfer 22.5 µL of master mix to each well of the appropriate PCR plate.
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for 00:00:10 , and spin down briefly in a plate centrifuge (00:00:05 at 2500 rpm ).
Amplify samples using the following PCR conditions:
98 °C for 00:00:30
35 cycles of:
98 °C for 00:00:15
65 °C for 00:05:00
Indexing PCR
Indexing PCR
For each sample, combine 10 µL of each of the four pools in a single Bio-Rad fully-skirted 96 well plate.
Seal plate with a “F” seal, mix well by vortexing using a plate vortexer at 1900 rpm for 00:00:10 , and spin down in a plate centrifuge (00:00:30 at 2500 rpm ).
In a 96-well Thermo plate, add 2 µL of each sample to 198 µL of nuclease free water (1:100 dilution).
Seal plate with a “F” seal, mix well by vortexing using a plate vortexer at 2500 rpm for 00:00:10 , and spin down in a plate centrifuge ( 00:00:30 at 2500 rpm ).
Transfer 5 µL of 1:100 diluted PCR 1 sample to a 96-well Thermo PCR plate.
Transfer 2 µL of 5 µM indexing primer mix to the 96-well Thermo PCR plate containing the samples.
Set up the following PCR master mix (multiply below volumes by number of reactions plus desired overage):
0.7 µL nuclease-free water
2 µL 5x Q5 reaction buffer
0.2 µL 10 mM dNTPs
0.1 µL Q5 Polymerase
Transfer 3 µL of master mix to each well of the appropriate PCR plate.
Seal plate with a “B” seal, mix well by vortexing using a plate vortexer at 1900 rpm for 00:00:10 , and spin down briefly in a plate centrifuge (00:00:05 at 2500 rpm ).
Amplify samples using the following PCR conditions:
98 °C for 00:00:30
10 cycles of:
98 °C for 00:00:20
55 °C for 00:00:15
72 °C for 00:01:00
72 °C for 00:05:00
Normalization
Normalization
Normalize samples using a SequalPrep plate according to manufacturer’s instructions.
sequalprep_platekit_man.pdf
sequalprep_platekit_man.pdf Elute in 20 µL of SequalPrep Elution Buffer.
Pooling
Pooling
Pool 10 µL of each sample in a trough, mix well and transfer material to a 1.5 mL non-stick tube.
Purify using AMPureXP beads at a 0.7x ratio. Elute library in 20 µL of EB.
Library QC
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
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.