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.
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.
|Sample type||Bronchoalveolar lavage (BAL)|
|Mean coverage (x)||440.7|
|Aligned reads (count)||96,059|
|Aligned reads (%)||99.5|
|SNP calls (count)||10|
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.
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.
|1. Purify Total RNA||MagMAX Viral/Pathogen Nucleic Acid Isolation Kit||A48310, A42352|
|2. Reverse transcribe RNA into cDNA||SuperScript IV VILO Master Mix
Second Strand cDNA Synthesis Kit
|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
Qubit fluorometric assays
|qPCR A38524100, A38524500
Qubit Q32850 Q32853
Optimized protocol to accelerate study of SARS-CoV-2 samples
Step 1: Purify total RNA
Step 2: Reverse transcribe RNA into cDNA
Step 3: Prepare NGS libraries
The following modifications are recommended to the Collibri ES DNA Library Prep Kit for Illumina Systems standard protocol.
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.|
|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.|
|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|
|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.
|SARS-CoV-2 genomic evolution||Following library prep, enrich for SARS-CoV-2||
|Host-pathogen interactions||No enrichment. Proceed to library quantification||
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
|Final extension||1 cycle||72°C||1 minutes|
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.
All sequencing methods
For Research Use Only. Not for use in diagnostic procedures.