Optimized Protocol for Collibri Next-Generation Sequencing of SARS-CoV-2

This hypothesis-free, optimized protocol for Invitrogen Collibri Library Prep Kits for Illumina™ Systems to sequence SARS-CoV-2 samples provides strong coverage and sensitive variant detection. It is compatible with all Illumina NGS systems.

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Sequence SARS-CoV-2 to monitor transmission and evolution

Next-generation sequencing (NGS) of the SARS-CoV-2 virus can enable monitoring of global transmission and lead to insights into viral evolution and pathology. The hypothesis-free analysis of SARS-CoV-2 genomes provided by Collibri DNA Library Prep Kits for Illumina Systems provides high coverage with sensitive variant detection (Figure 1). Faster, sensitive analysis of emerging strains allows the research community to provide insights for vaccine and therapeutic development.

The workflow instructions below are optimized for the study of coronaviruses, including SARS-CoV-2, on Illumina NGS systems. Lysates from bronchoalveolar lavage (BAL) research samples obtained with consent from the Santara Clinics Biobank in Lithuania were purified and reverse transcribed using a SuperScript IV VILO Master Mix and Thermo Scientific Second Strand cDNA Synthesis Kit. Resulting cDNA was converted into NGS libraries using Collibri ES DNA Library Prep Kits for Illumina Systems and further enriched. Libraries were sequenced 2 x 150 bp on an Illumina MiSeq™️ System.

NGS coverage of SARS-CoV-2 samples from Collibri library prep kits Strong coverage from low sequencing of SARS-CoV-2 libraries using the Collibri library prep kits

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	Sample 1
Sample type	Bronchoalveolar lavage (BAL)
Mean coverage (x)	440.7
Aligned reads (count)	96,059
Aligned reads (%)	99.5
SNP calls (count)	10

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	Sample 2
Sample type	Bronchoalveolar lavage (BAL)
Mean coverage (x)612	612.1
Aligned reads (count)	134,431
Aligned reads (%)	99.6
SNP calls (count)	12

Figure 1. Strong coverage and sensitive variant detection. Coverage profiles from two research samples obtained from patients who tested positive for COVID-19 demonstrate coverage of the entire genomes from less than 250,000 reads per sample. Variant detection sensitivity is suitable for strain identification of individual samples.

Workflow

Purification

DNA synthesis

Library generation

Enrichment (optional)

Library quantification & sequencing

Required materials to sequence SARS-CoV-2

The optimized protocol for studying SARS-CoV-2 by NGS on Illumina systems uses the reagents shown in the table below.

Table 1. Reagents for each step of the optimized protocol to sequence SARS-CoV-2 are fully supported by Thermo Fisher Scientific.

Step	Kit	Catalog numbers
1. Purify Total RNA	MagMAX Viral/Pathogen Nucleic Acid Isolation Kit	A48310, A42352
2. Reverse transcribe RNA into cDNA	SuperScript IV VILO Master Mix and Second Strand cDNA Synthesis Kit	11756050, 11756500 and A48570, A48571
3. Prepare NGS libraries	Collibri ES DNA Library Prep Kit for Illumina Systems	Combinatorial Dual indexes (CD) A38605024, A38607096 Unique Dual Indexes (UD) A38606024, A43605024, A43606024, A43607024
4. Quantify libraries	Collibri Library Quantification Kit or Qubit fluorometric assays	qPCR A38524100, A38524500 or Qubit Q32850 Q32853

Optimized protocol to accelerate study of SARS-CoV-2 samples

Step 1: Purify total RNA

After lysing the BAL sample, purify total RNA. The MagMAX Viral/Pathogen Nucleic Acid Isolation Kit has been optimized to increase SARS-CoV-2 testing throughput.

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Step 2: Reverse transcribe RNA into cDNA

For best results, generate first strand cDNA using SuperScript IV VILO Master Mix and generate second strand cDNA using Second Strand cDNA Synthesis Kit.

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Download Second Strand cDNA Synthesis Kit user guide

Step 3: Prepare NGS libraries

The following modifications are recommended to the Collibri ES DNA Library Prep Kit for Illumina Systems standard protocol.

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Table 2. Recommended protocol optimizations for library generation using Collibri ES DNA Library Prep Kits.

Step	Standard recommendation			Recommended changes for SARS-CoV-2 samples
1. Remove EDTA from DNA samples	(if needed)			Begin with 25 µL after completing reverse transcription
	Input: 1–500 ng			Input: 50 ng
2. Fragment the DNA and add dA-tails	On ice or a cooling rack, assemble the fragmentation and dA-tailing reaction for each DNA sample in a sterile 0.2-mL thin-wall PCR tube. Add the reagents in the order given.			On ice or a cooling rack, assemble the fragmentation and dA-tailing reaction for each DNA sample in a sterile 0.2-mL thin-wall PCR tube. Add the reagents in the order given.
	Component	Volume		Component	Volume
	10 mM Tris-HCL, ph 7.5-8.5	to 40 µL		cDNA	10 µL
	Double-stranded DNA (1 ng - 500 ng)	X µL		10 mM Tris-HCI, pH 7.5-8.5	to 31 µL
	10X Fragmentation and dA-tailing Buffer (blue )	5 µL		10X Fragmentation and dA-tailing Buffer (blue)	5 µL
	Total volume (light blue mixture )	40 µL		Total volume (light blue mixture)	36 µL
	Add 5X Fragmentation and dA-tailing Enzyme Mix to the sample.			Add 5X Fragmentation and dA-tailing Enzyme Mix to the sample.
	Component	Volume		Component	Volume
	Buffer-DNA mixture from step 1 (light blue mixture)	40 µL		Buffer-DNA mixture from step 1 (light blue mixture)	36 µL
	5X Fragmentation and dA-tailing Enzyme Mix (clear)	10 µL		5X Fragmentation and dA-tailing Enzyme Mix (clear)	14 µL
	Total volume (light blue mixture)	50 µL		Total volume (light blue mixture)	50 µL
	Table 2 Recommended fragmentation time and optimization range to attain the desired fragment size			Fragment for 20 minutes at 37℃
	Fragment size	Fragmentation time at 37°C
	Fragment size	Recommen-dation	Optimization range
	150-300 bp	20 min	20-30 min
	300-500 bp	10 min	10-20 min
	500-700 bp	5 min	5-10 min
3. Carry out post-ligation double-sided size selection	Double-sided size selection to match target insert size			Customized cleanup protocol
4. PCR amplify the library	The number of PCR cycles depends on the starting amount of DNA (i.e., input DNA).			Amplify the library for 12 PCR cycles.

Step 4: (Optional) Enrich libraries for SARS-CoV-2

This optional enrichment step is recommended for projects with a focus on coronavirus genomes. This step may be omitted for projects that study the interaction between viral genomes and their hosts. If enrichment is performed, additional amplification is recommended to ensure maximum yields (Table 4).

Table 3. Decision to perform optional enrichment depends upon project goals.

Research goal	Recommendation	Benefits
SARS-CoV-2 genomic evolution	Following library prep, enrich for SARS-CoV-2	Enhanced percentage of reads mapping to SARS-CoV-2 Reduced cost of sequencing
Host-pathogen interactions	No enrichment. Proceed to library quantification	Retain sequences of both host and SARS-CoV-2

Amplify enriched libraries

Eight PCR cycles are performed in a thermal cycler with the lid temperature set to 105°C using the Collibri 2X library amplification master mix. After the PCR is completed, proceed with the post-amplification cleanup (see "Purify the amplified DNA libraries” on page 27 of the Collibri ES DNA Library Prep Kit for Illumina Systems manual).

Table 4. Recommended PCR conditions to amplify enriched NGS libraries.

Stage	Number of cycles	Temperature	Time
Activate the enzyme	1 cycle	98°C	30 seconds
Denature	3-4 cycles for 100 ng of input DNA 6-8 cycles of 10 ng of input DNA 10-12 cycles for 1 ng of input DNA	98°C	15 seconds
Anneal		60°C	30 seconds
Extend		72°C	30 seconds
Final extension	1 cycle	72°C	1 minutes
Hold	1 cycle	4°C	Hold

Step 5: Quantify libraries and sequence

Determine concentration of final sequencing library using the Collibri Library Quantification Kits. No modifications recommended. Proceed to load the flow cell as recommended by Illumina. Libraries are compatible with all Illumina NGS systems.