An optimized, amplicon-based approach for sequencing of SARS-CoV-2 from patient samples using COVIDSeq assay on Illumina MiSeq sequencing platforms.

Sequencing of SARS-CoV-2 genomes is crucial for understanding the genetic epidemiology of the COVID-19 pandemic. It is also critical for understanding the evolution of the virus and also for the rapid development of diagnostic tools. The present protocol is a modification of the Illumina COVIDSeq test. We describe an amplicon-based next-generation sequencing approach with short turnaround time, adapted for bench-top sequencers like MiSeq, iSeq, and MiniSeq. For complete details on the use and execution of this protocol, please refer to Bhoyar et al. (2021).

Before you begin

The study was approved by the Institutional Human Ethics Committee (IHEC No. Dated CSIR-IGIB/IHEC/2020-21/01). After obtaining informed consent from patients, samples from nasal, nasopharyngeal, and oropharyngeal swabs were collected according to the standard protocol in 3 mL sterile viral transport medium (VTM) tube. All the samples were transported to the laboratory at a cold temperature (2°C–8°C) within 72 h post collection, and stored at −80°C till further used.

Introduction

The Illumina COVIDSeq assay is an amplicon-based next-generation sequencing (NGS) approach for the detection of SARS-CoV-2 RNA isolated from nasopharyngeal, oropharyngeal, and mid-turbinate nasal swabs from patients. The assay is one of the first NGS based SARS-CoV-2 detection assays approved for use under U.S Food and Drug Administration’s Emergency Use Authorization (EUA). The COVIDSeq test was first standardized and validated for Illumina NovaSeq sequencer and can accommodate 384 to 3072 samples, depending on the configuration of flow cells and the instrument (Bhoyar et al. 2021). RNA required for SARS-CoV-2 sequencing can be extracted from decontaminated nasopharyngeal swabs using the methods mentioned in EUA approval that include; Quick-DNA/RNA Viral MagBead Kit (Zymo Research, # R2141) or QIAamp Viral RNA Mini Kit (Qiagen, part # 52906). However, the protocol is compatible with all commonly used viral RNA isolation methods including, Nextractor® NX-48S (GENOLUTION), Trueprep (Molbio Diagnostics Pvt. Ltd.) and TRIzol (Invitrogen).

While originally adapted and optimized for larger sequencers, this assay could be optimized for low throughput bench-top equipment for smaller batch sizes. This manuscript details the optimized protocol with marginal modifications along with an open-source pipeline for the analysis and interpretation of data. While the protocol has been standardized for the Illumina MiSeq sequencing platform, by considering the final loading concentration of the library and the data output, the proposed protocol can be adopted for any bench-top sequencer from Illumina Inc.

Requirements for library preparation

This protocol consists of the following key steps:

RNA extraction- Prior to RNA extraction, the VTM samples were subjected to heat inactivation at 50°C for 30 min. RNA is extracted from the inactivated specimen (VTM), using any of the following mentioned protocol that includes; QIAmp (QIAGEN), Nextractor® NX-48S (GENOLUTION), TruePrep (Molbio Diagnostics Pvt. Ltd.) and TRIzol (Invitrogen).

cDNA synthesis- Generates complementary DNA to the RNA by reverse transcriptase using random hexamers.

Target amplification- The synthesized cDNA undergoes two separate PCR reactions to amplify the virus genome present in the sample.

Library preparation, Pooling and Quantification- During this process, the pooled amplified products undergo bead-based tagmentation where it gets fragmented and tagged to the adapter sequences. The adapter-tagged fragments undergo another round of PCR amplification, after which indexed tagged libraries will be pooled and cleaned using the purification beads. The pooled library product is quantified using Qubit High Sensitivity dsDNA quantification kit (Invitrogen).

Sequencing- The sequencing-ready libraries are clustered onto a flow cell and sequenced using sequencing by synthesis (SBS) chemistry on the Illumina MiSeq sequencing system.

Analysis- This step includes genome assembly, variant calling and phylogenetic analysis using established protocols (available in the GitHub repository:https://github.com/bnijolly/Genepi).

Prepare the following reagents

Isolated SARS-CoV-2 RNA samples.

A pre-amplification environment with a Biosafety level 2 (BSL-2) facility is required during RNA sample handling and cDNA preparation.

Personal Protective Equipment (PPE), as recommended by WHO for handling viral samples contagious to humans.

Two 96-well PCR thermal cyclers.

Thermo mixer with a 96-well PCR plate holder.

Qubit HS dsDNA quantification kit along with instrument (Thermofisher).

cDNA preparation

Timing: 1 h

During this process, the extracted SARS-CoV-2 RNA is annealed using random hexamers and the RNA fragments primed with random hexamers are reverse-transcribed into first strand cDNA using reverse transcriptase enzyme.

(All steps to be carried out within the Biosafety cabinet of the BSL-2 facility)

Annealing of RNA

Label a new 96 well PCR plate as cDNA plate (Figure 1).

Figure 1

Layout of the sample plate to be used denoting the samples, PC and NC

Up-to 94 samples can be processed at once. A positive control (CPC HT) and Negative Control (ELB HT)is included in every plate that is processed using the COVIDSeq protocol.

Add 8.5 μL of the isolated SARS-CoV-2 sample into the wells of the cDNA plate.

Add 8.5 μL of EPH3 HT to each well.

Mix well with a P10 or P20 multichannel pipette. If plate shaker is available, within the laminar hood, shake the plate at 1600 rpm for 1 min.

Seal and spin the plate at 1000 × g for 1 min.

Place the cDNA plate on a thermal cycler and run the following program

Choose the preheat lid option

Set the final volume of the reaction as 17 μL

65oC for 3 min

Hold at 4oC

First strand cDNA synthesis

Prepare First strand cDNA Mastermix (for 96 samples) by mixing 864 μL of FSM HT and 96 μL of RVT HT in a 1.5mL microcentrifuge tube.

Add 8 μL of the First strand cDNA mastermix into each well of the cDNA plate and pipette well to mix.

Seal and spin the plate at 1000 × g for 1 min.

Place the cDNA plate on a thermal cycler and run the following program

Choose the preheat lid option

Set the final volume of the reaction as 25 μL

25oC for 5 min

50oC for 10 min

80oC for 5 min

Hold at 4oC

Pause point: The procedure can be paused here and the cDNA plate can be stored at −20oC for up to 7 days.

Amplification of cDNA

Timing: 4 h

In this step, the SARS-CoV-2 virus genome present in the test samples is amplified using two separate PCR reactions that are then pooled together.

Amplification of prepared cDNA

Label two 96-well PCR plates as COV1 and COV2.

Prepare PCR1 Mastermix and PCR2 Mastermix in two separate 15 mL tubes.

Add the following to each tube:

Add 5 μL each of first strand cDNA from the cDNA plate to corresponding wells of COV1 and COV2 plates.

Add 20 μL of PCR1 Mastermix to each well of COV1 plate.

Add 20 μL of PCR2 Mastermix to each well of COV2 plate.

Seal and shake the COV1 and COV2 plates at 1600 rpm for 1 min.

Centrifuge the plates at 1000 × g for 1 min.

Place the plates on individual thermal cyclers and run the following program

Choose the preheat lid option

Set the final volume of the reaction as 25 μL

Set up the PCR with the following conditions

The program has a run time of approximately 03 h and 30 min.

Pause point: The procedure can be paused here and the COV1 and COV2 plates can be stored at −20oC for up to 3 days.

Key resources table

Materials and equipment

Step-by-step method details

Library preparation and library pooling

Timing: 3–4 h

In this step, the prepared cDNA of the SARS-CoV-2 genome is tagmented (tagged and fragmented), indexed and amplified to become sequencing-ready libraries.

Tagmentation of PCR amplicons

Label a new 96-well PCR plate as TAG plate.

From COV1 and COV2 plates, combine 10 μL of contents from each well to their respective wells in TAG1 plate, and mix well. This results in a total volume of 20 μL in each well of the TAG1 plate.

To make Tagmentation Mastermix:

Label a 15 mL tube as Tagmentation Mastermix.

Follow the table below to prepare Tagmentation Mastermix.

Vortex for 30 s to mix well.

Add 30 μL of Tagmentation Mastermix to each well in the TAG1 plate.

Seal and shake the TAG1 plate at 1600 rpm for 1 min.

Centrifuge at 1000 × g for 1 min if the contents of the well are found sticking to the walls or the seal of the TAG1 plate.

Place on a thermal cycler and run the following program.

Choose the preheat lid option

Set the total volume of the reaction as 50 μL.

55oC for 5 min

Hold at 10oC.

Post Tagmentation Clean-up

Centrifuge the TAG1 plate at 500 × g for 1 min.

Remove the seal and add 10 μL of ST2 HT to each well of the TAG1 plate.

Seal and shake at 1600rpm for 1 min.

Incubate the plate at room temperature (22°C–25°C) for 5 min.

Centrifuge the TAG1 plate at 500 × g for 1 min.

Remove the seal carefully and place the plate on a magnetic stand.

Wait for 3 min/until the liquid is clear.

Remove and discard all supernatants from the wells.

Wash the beads as follows

Remove the plate from the magnetic stand.

Add 100 μL of TWB HT to each well.

Seal and shake at 1600 rpm for 1 min and centrifuge at 500 × g for 1 min.

Remove the seal and place the plate on the magnetic stand. Wait for 3 min/until the liquid is clear.

Remove and discard the supernatant from each well.

Repeat the wash steps (step i; i-v) one more time.

CRITICAL: Do not discard the supernatant after the second wash, to prevent the beads from over drying.

Amplification of tagmented amplicons

In a 15 mL tube, prepare Enhanced PCR Mix

Add 2304 μL of EPM HT (24 μL × 96 samples).

Add 2304 μL of Nuclease-free water (24 μL × 96 samples).

Vortex the tube to mix.

Remove and discard the supernatant from the TAG1 plate.

Use a 10 μL or 20 μL pipette to remove any remaining TWB HT from the TAG1 plate.

Add 40 μL of Enhanced PCR Mix to each well of the TAG1 plate.

Add 10 μL of index adapters to each well of the TAG1 plate.

Seal and shake the plate at 1600 rpm for 1 min and centrifuge the plate at 500 × g for 1 min.

CRITICAL: Inspect the tubes to make sure the beads are fully resuspended and are not found at the bottom of the tubes.

Place on a thermal cycler and run the following PCR program

Choose the preheat lid option and set the temperature to 100oC.

Set the total volume of the reaction to be 50 μL.

Library pooling and Clean up

Centrifuge the TAG1 plate at 500 × g for 1 min.

Remove the seal and place the plate on a magnetic stand and wait for 3 min/until the liquid is clear.

Transfer 5 μL of library from each well into an 8-tubes strip. This results in a pooled volume of 60 μL in each tube of the 8-tubes strip.

CRITICAL: Discard and change tips for each column of samples while transferring.

Transfer 55 μL of pooled library from each tube to a 1.5 mL microcentrifuge tube. This results in a total of 440 μL of pooled libraries (from 96 samples).

Add 396 μL of ITB to each tube (ITB volume is calculated by multiplying the total volume of the pooled libraries with 0.9 i.e., for 96 samples: 440 μL × 0.9 = 396 μL)

Vortex the tube to mix well.

Incubate the tube at room temperature for 5 min.

Spin briefly and place the tube on a 1.5mL tube magnetic stand and wait for 5 min.

Remove and discard all supernatants.

Wash the beads as follows:

Add 1000 μL of freshly prepared 80% ethanol to the tube and wait for 30 s.

Remove and discard the supernatant.

Repeat the above-mentioned wash steps one more time for a total of 2 washes with 80% ethanol.

Remove any residual ethanol left on the tube.

Air dry the beads for 2 min.

Add 55 μL of Resuspension Buffer to the tube and vortex to mix.

Incubate at room temperature for 2 min.

Spin briefly and place the tube in the magnetic stand and wait for 2 min.

Label a new 1.5 mL tube as Final Pool.

Transfer 50 μL of the supernatant into the Final Pool tube.

Pause point: The procedure can be paused here and the Final Pool tube can be stored at −20oC for 30 days.

Quantification and Normalization of the library pool

Using the Qubit High Sensitivity dsDNA quantification kit (Invitrogen), quantify the library pool

Dilute 1 μL of the pooled library sample in the ratio of 1:3.

Use 2 μL of the diluted library for quantification.

Calculate the actual concentration of the library by factoring in the dilution.

Load 2 μL of the pooled library (undiluted) onto a 2% agarose gel. The libraries should be observed between the size 300–400 bp.

Using the formula below, calculate the concentration of the library in Molar units.

Dilute the library with Resuspension Buffer to a final concentration of 4nM.

The formula for calculating Molarity:

[Library Conc (ng/μL)]/ [660(g/mol) x average library size (bp)]×106= Conc. of Library (nM)

Sequencing the libraries

Timing: ~22 h: according to MiSeq reagent kit V3 (2 × 75bp)

The prepared COVIDSeq libraries will be sequenced in this step using the MiSeq sequencing platform

Preparing PhiX

Label a new 1.5 mL tube as PhiX Control.

Combine 2 μL of PhiX stock library (10nM) and 3 μL of Nuclease-free water to make 4nM PhiX library

CRITICAL: Prepare a fresh PhiX dilution and use it within 12 hours of preparation.

Label a new 1.5 mL tube as PhiX-Final and add 5 μL of diluted PhiX (4nM) and 5 μL of 0.2N freshly prepared NaOH.

Vortex the tube briefly to mix. Spin and incubate the tube at room temperature for 5 min.

Add 990 μL of prechilled HT1 to the PhiX-Final tube resulting in 1mL of 20pM PhiX library.

Denaturation of COVIDSeq library

Label a new 1.5 mL tube as Library-Final and add 5 μL of diluted library (4nM) and 5 μL of 0.2N freshly prepared NaOH.

Vortex the tube briefly to mix. Spin and incubate the tube at room temperature for 5 min.

Add 990 μL of pre-chilled HT1 to the tube resulting in 1mL of 20pM Final library.

CRITICAL: For instructions regarding final loading concentration and PhiX spike-in for MiSeq system, refer to the MiSeq system Denature and Dilute Libraries Guide.

To prepare 6pM final loading concentration library

Label a new 1.5mL tube as 6pM Final Library tube.

Add 180 μL of the 20pM library from the Library-Final tube.

Add 420 μL of pre-chilled HT1 and mix well.

Discard 6 μL of the mixture.

Add 6 μL of the 20pM PhiX library for 1% spike in as recommended by Illumina Inc.

Mix well with a pipette, spin briefly and place the tube on ice. The library is ready to be loaded onto the flow cell.

CRITICAL: The final library has to be loaded onto the flow cell within 30 minutes of preparation.

To reduce the handling time, the denaturation of the library and PhiX can be performed simultaneously.

Sequencing and expected Output

Load the library into the MiSeq Sequencing cartridge and initiate sequencing as per the MiSeq Sequencing guide by Illumina Inc.

For sequencing using MiSeq v3 2×75 cycles kit, the estimated yield is about 3.3–3.8 Gb as per Illumina Inc.

Expected outcomes

Upon successful tagmentation and size selection the final library pool will appear as ~300 bp size band on 2% agarose gel (Figure 2). Any deviation in the fragment size may be the result of erroneous tagmentation or size selection, which can be minimized with proper handling and pipetting practices.

Figure 2

Quality of the final library pool was analyzed by agarose gel electrophoresis (2%), the expected fragment of ~300 bp size was observed

Quantification and statistical analysis

Genome assembly, variant calling, and phylogenetic analysis

This step describes the basic bioinformatics pipeline for genome assembly and identification of variants in the SARS-CoV-2 genome. The raw sequencing data generated by Illumina sequencing platforms are in the form of Binary Base Call (BCL) files and require conversion to FASTQ format before further processing.

The BCL files generated by the sequencer are demultiplexed to FASTQ files using the bcl2fastq conversion software provided by Illumina using the command:

bcl2fastq --runfolder-dir --sample-sheet --output-dir

The raw FASTQ files need to undergo additional processing steps, including quality control, trimming, alignment against the SARS-CoV-2 reference genome and sorting. The resulting BAM file containing mapped and sorted reads are then used to call variants and generate a consensus sequence of the genomes in FASTA file format. The steps to assemble SARS-CoV-2 genomes from raw FASTQ files are detailed in a recent protocol (Poojary et al 2020). Sample parameters and metrics that may be used to assess the quality of the assembled genomes are given in Table 1.

Table 1

Data generated from a single sample of the 96 samples sequenced using the MiSeq platform

Sample ID	Number of raw reads R1	Number of raw reads R2	Number of reads after trimming R1	Number of reads after trimming R2	% Of reads Aligned with reference	Number of base Pairs Covered	X coverage	Genome coverage	Number of variants called	Pango lineage	Time taken
CSXXXX	1431919	1431919	1221580	1221580	99.52%	84310380	2819.46	99.84%	11	B.1.36	318 s

For assignment of lineages to the assembled genome FASTA sequences, we use the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) software command line tool using the command:

pangolin .fasta

Alternatively, the web version of PANGOLIN (available at https://pangolin.cog-uk.io/) can be used to assign lineages to the generated FASTA sequences (Rambaut et al 2020).

The FASTA sequence files can also be used to analyze and visualize evolutionary relationships through phylogenetic clustering. For performing the phylogenetic analysis, we use the open-source tool-kit Nextstrain following a recently detailed protocol (Jolly and Scaria 2021). The comparison of data output, coverage and time taken for analysis between MiSeq and NovaSeq 6000 is provided in the Tables S1 and S2.

Limitations

As explained in our previous report (Bhoyar et al 2021), the Illumina COVIDSeq assay can detect RNA from SARS-CoV-2 in nasopharyngeal, oropharyngeal, and mid-turbinate nasal swabs from patients suspected to have COVID-19 with the highest CT 37 to the lowest CT samples. However, we have noticed that in order to capture the whole SARS-CoV-2 genome, RNA samples below CT 22 should be considered.

Troubleshooting

Problem 1

Amplification in the negative control (NC) (Step: cDNA Preparation)

In some cases, users may get amplification in the negative control well in the COVIDSeq assay.

Potential solution

To avoid amplification in the negative control we encourage the users to stick to the following practices.

Autoclave all the pipettes before initiating a new experiment.

Thoroughly decontaminate the Biosafety cabinet before the experiment.

Use fresh pipette tips and fresh consumables between samples and dispensing reagents.

Use aerosol resistant pipette tips to minimize the risk of carry over and sample to sample cross contamination.

Use fresh PCR plate sealing films every time.

Problem 2

Low genome coverage in samples (Step-Genome Assembly, Variant Calling and Phylogenetic Analysis).

Users may find the genome coverage in certain samples is low and the samples do not return any variant after the analysis.

Users may ensure that the starting material has a high viral load i.e., we recommend samples with a CT value of <25 for 99% genome coverage.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Sridhar Sivasubbu (sridhar@igib.in).

Materials availability

All the materials used in the protocol are available without any restrictions; the details of the kits used in the protocol are given in the “key resources table”.

Data and code availability

All the scripts used for the SARS-CoV-2 sequencing analysis and interpretation are adopted from Poojary et al 2020 (GitHub repository: https://github.com/banijolly/Genepi).

S.No	Item name	Volume for 96 samples	Volume for 1 sample
1	Nuclease Free Water	451.2 μL	4.7 μL
2	IPM HT	1440 μL	15μL
3	CPP1 HT (for PCR1)/ CPP2 HT (for PCR2)	412.8μL	4.3μL

PCR cycling conditions
Steps	Temperature	Time	Cycles
Initial Denaturation	98oC	3 min	x 1 cycle
Denaturation	98oC	15 s	x 35 cycles
Annealing	65oC	5 min
Hold	4oC	∞

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ethanol, Molecular Grade	Merck	100983
Nuclease-Free Water	Ambion	AM9906
Critical commercial assays
Illumina COVIDSeq Test 3072 samples	Illumina	20043675
MiSeq Reagent Kit V3 (150 cycles)	Illumina	MS-102-3001
Qubit dsDNA HS Assay Kit	Thermo Fisher	Q32851
Biological samples
SARS-CoV-2 RNA	NasopharyngealoOropharyngeal swab samples	NA
Oligonucleotides
IDT for Illumina PCR Indexes Set 1–4	Illumina	20043137
Software and Algorithms
bcl2fastq Conversion Software	Illumina	NA
Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN)	Center for Genomic Pathogen Surveillance	https://github.com/cov-lineages/pangolin
Nextstrain open-source toolkit	Hadfield et al.,2018	https://github.com/nextstrain
Deposited data
Scripts used for the SARS-CoV-2 sequencing analysis and interpretation	Poojary et al., 2020	https://github.com/banijolly/Genepi
Other
96-Well PCR thermal cyclers	Veriti 96-Well Thermal Cycler	4375786
Thermomixer with a 96-well PCR plate holder	Eppendorf ThermoMixer C	05–412-503
96-Well PCR plate	Thermo Fisher	AB0600

Reagent	Description	Storage	Total volume required per reaction
SARS-CoV-2 RNA a	Sample	−20oC	8.5 μL
CPC HTb	COVIDSeq Positive Control HT	−20oC	8.5 μL
ELB HTb	Elution Buffer HT	−20oC	8.5 μL
EPH3 HT	Elution Prime Fragment 3HC Mix	−20oC	8.5 μL
FSM HT	First Strand Mix HT	−20oC	9 μL
RVT HT	Reverse Transcriptase HT	−20oC	1 μL
IPM HT	Illumina PCR Mix HT	−20oC	15 μL
CPP1 HT	COVIDSeq Primer Pool HT 1	−20oC	4.3 μL
CPP2 HT	COVIDSeq Primer Pool HT 2	−20oC	4.3 μL
TB1 HT	Tagmentation Buffer 1 HT	4oC	12 μL
EBLTS HT	Enrichment BLT HT	−20oC	4 μL
ST2 HT	Stop TagmentBuffer2 HT	4oC	10 μL
TWB HT	Tagmentation Wash Buffer HT	4oC	200 μL
EPM HT	Enhanced PCR Mix HT	−20oC	24 μL
Index adaptera	IDT for Illumina PCR Indexes- Set 1,2,3,4	−20oC	10 μL
ITB	Illumina Tune Beads	RT	396 μL (for 96 samples)
80% Ethanola	80% Ethanol	RT	2 mL
RSB	Resuspension Buffer	4oC	55 μL
0.2N NaOH	Denaturation solution	RT	10 μL
HT1	Neutralizing Buffer	−20oC	2.4 mL
PhiX	Spike in Control	20oC	5 μL
NFWa	Nuclease Free Water	RT	25 mL

Items not included along with the Illumina COVIDSeq test kit

Positive control and Elution Buffer are provided in the COVIDSeq test kit. CPC HT is provided at a stock concentration of 1 million copies/μL. A working solution of 5 copies/μL have to be prepared before adding to the plate. Elution buffer is used directly as a negative control.

S.No	Item name	Volume for 96 samples	Volume for 1 sample
1	Nuclease-free water	1920 μL	20 μL
2	TB1 HT	1152 μL	12 μL
3	EBLTS HT	384 μL	4 μL

PCR cycling conditions
Steps	Temperature	Time	Cycles
Initialization	72oC	3 min	x1 cycle
Initial Denaturation	98oC	3 min	x1 cycle
Denaturation	98oC	20 s	x7 cycles
Annealing	60oC	30 s
Extension	72oC	1 min
Final extension	72oC	3 min	x1 cycle
Hold	10oC	∞