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Optimized Protocol for Collibri Next-Generation Sequencing of SARS-CoV-2
Next-generation sequencing (NGS) of the SARS-CoV-2 virus can enable surveillance of global transmission and lead to insights into viral evolution and pathology. The analysis of SARS-CoV-2 genomes provided by Collibri DNA Library Prep Kits for Illumina Systems provides high coverage with sensitive variant detection. Faster, sensitive analysis of emerging strains allows the research community to provide insights for vaccine and therapeutic development. It is compatible with all Illumina NGS systems.
With the continuous rise of new SARS-CoV-2 variants, there's a need for solid, precise, and quick sequencing strategies. The workflow instructions below are optimized for the study of coronaviruses following amplicon sequencing protocol originally created by ARTIC Network.
We currently support two protocols with PCR enrichment:
1. Recommended protocol: A workflow using SuperScript IV Vilo Master Mix and Phusion(TM) Plus Master Mix for cDNA synthesis and PCR enrichment, respectively.
2. Alternative protocol: A workflow using SuperScript IV RT and Platinum SuperFi U MasterMix for cDNA synthesis and PCR enrichment, respectively.
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
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 | 11756050, 11756500 |
| 3. Enrich PCR | ARTIC v3 primer pools Phusion Plus PCR Master Mix | Check the ARTIC Network website for the latest release. F631XL, F631L |
| 4. Prepare NGS libraries | Collibri ES DNA Library Prep Kit for Illumina Systems *The kit contains adapters with unique dual indexes (UDs) or combinatorial dual indexes (CDs). | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD) to support up to 192 preparations per run. See Table 7 for more recommendations. |
| 5. Quantify libraries | Collibri Library Quantification Kit or Qubit dsRNA HS Assay Kit | qPCR (recommended method) A38524100, A38524500 or Qubit Q32851, Q32854 |
Step 1: Perform RNA purification
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.
Step 2: Reverse transcribe RNA into cDNA
Generate cDNA using SuperScript IV VILO Master Mix according to the following recommendations:
Table 2. Recommendations for reaction set-up and cycling for cDNA generation.
| Component | Volume 10 µL rxn |
|---|---|
| Water, nuclease free | 2.5 µL |
| SuperScript IV VILO Master Mix | 2 µL |
| Purified RNA | 5.5 µL |
| Temperature | Time |
|---|---|
| 25ºC | 10 min |
| 50ºC | 10 min |
| 85ºC | 5 min |
Step 3: Perform PCR enrichment
Perform PCR enrichment (two reactions by sample) using the ARTIC primer pool according to the following recommendations.
Table 3. Reaction set-up for enriching viral sequences
| Component | Volume, 25 µL rxn |
|---|---|
| Water, nuclease free | 9 µL |
| 2X Phusion Plus PCR Master Mix | 12.5 µL |
| Primer Pool (1 or 2) | 1 µL |
| cDNA from previous reaction | 2.5 µL |
Check the ARTIC Network website for the ARTIC primer pool latest release.
Table 4. Cycling parameter for PCR enrichment
| Temperature | Time | Cycles |
|---|---|---|
| 98ºC | 30 sec | 1 |
| 98ºC | 15 sec | 30 |
| 63ºC | 5 min | |
| 4ºC | hold | 1 |
Combine 12.5 µL of each pool (25 µL in total) and perform PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide.
Step 4: Prepare NGS libraries
The following modifications are recommended to the Collibri ES DNA Library Prep Kit for Illumina Systems standard protocol.
- 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.
Table 5. Fragmentation and dA-tailing reactions - Incubate for 15 minutes at 37°C followed by 10 minutes at 65°C.
- Add 10 µL of the 7X Ligation mix and 10 µL of the adapter mix each sample on ice, and incubate for 30 minutes at 20ºC.
- Perform the post-ligation purification with magnetic beads from Collibri ES DNA Library Prep Kit Pub. No. MAN0019527.
- Amplify the library according to the following recommendations.
- Purify the amplified libraries with the magnetic beads from Collibri ES DNA Library Prep Kit, analyzed by Agilent 2100 Bioanalyzer.
| Component | Volume |
|---|---|
| Purified DNA | 18 µL |
| Elution Buffer | 17 µL |
| 10X Fragmentation and dA-tailing Buffer (blue) | 5 µL |
| 5X Fragmentation and dA-tailing Enzyme Mix | 10 µL |
| Total volume (light blue mixture) | 50 µL |
Table 6. Library amplification reaction set-up and cycling.
| Component | Volume, 25 µl rxn |
|---|---|
| Purified DNA | 20 µL |
| 2X Library Amplification Master Mix | 25 µL |
| Primer mix | 5 µL |
| Temperature | Time | Cycles |
|---|---|---|
| 98ºC | 30 sec | 1 |
| 98ºC | 15 sec | 3 |
| 60ºC | 30 sec | |
| 72ºC | 30 sec | |
| 72ºC | 60 sec | 1 |
| 4ºC | Hold | 1 |
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.
Note: qPCR is the recommended QC method for NGS due to the technological advantage to discriminate between adapter having and lacing library fragments. We recommend using Collibri Library Quantification Kit
| Throughput | Recommendationn |
|---|---|
| 120 preparations per run | For mid-throughput with full Collibri kits, use A38605024 (CD) with A38607196 (UD) together or any UD set of 24 preparations with A38607096 (CD). |
| 192 preparations per run | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD). |
| >192 preparations per run | For unlimited throughput request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W). |
| Note: Do not pool together different sizes of kits containing the same type of indexed adaptors (CDs or UDs only). | |
| Note: A38606024, A38605024, A43606024, and A43607024 (24 preparations) together are equivalent to A38607196 (96 preparations) and can be ordered interchangeably. | |
Alternative protocol
The optimized Collibri Next-Generation Sequencing of SARS-CoV-2 protocol using PCR enrichment has been used to sequence a total of 1001 SARS-CoV-2 positive clinical samples. As evident in Figure 1, a targeted amplicon-based sequencing approach is not only cost-effective, but also provides high-quality results. Libraries were sequenced on the Illumina MiSeq system by paired-end sequencing of 2 x 150 bp, including a 10% v/v PhiX sequencing control. Alignment was performed with BWA (bwa mem -t 32 -r 1.0 -k 19 -M -B 6 -v 1). The mean coverage, or the average number of reads that align to the reference NCBI ASM985889v3 genome, has been calculated by QualiMap BamQC.
Collection 1 | Collection 2 | Collection 3 | Collection 4 | Collection 5 | Collection 6 | |
|---|---|---|---|---|---|---|
Number of samples | 93 | 192 | 192 | 192 | 140 | 192 |
Mean aligned reads (%) | 97.3 | 70.2 | 85.9 | 85.6 | 91.0 | 86.4 |
Mean aligned reads (counts) | 134,623 | 26,335 | 91,653 | 60,920 | 121,623 | 91,653 |
Figure 1. Percentage of samples with different depth of coverage within each of six collections. A total of 1001 SARS-CoV-2 positive clinical samples were sequenced. The results show that amplicon-based workflow solution using Collibri ES DNA Library Prep Kit for Illumina Systems enables population-wide viral surveillance and research.
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 Reverse Transcriptase and RNaseOUT Recombinant Ribonuclease Inhibitor and dNTP Mix (10 mM each) and Random Hexamer Primer | 18090050 and 10777019 and 18427013 and SO142 |
| 3. Enrich PCR | ARTIC v3 primer pools Platinum SuperFi U Multiplex Master Mix | Check the ARTIC Network website for the latest release. A5140024 or A5140096 |
| 4. Prepare NGS libraries | Collibri ES DNA Library Prep Kit for Illumina Systems | Combinatorial Dual indexes (CD) A38605024 (24 preparations), Unique Dual Indexes (UD) A38606024, A43605024, A43606024 and A43607024 (24 preparations) or See Table 3 for more recommendations. |
| 5. Quantify libraries | Collibri Library Quantification Kit or Qubit dsRNA HS Assay Kit | qPCR A38524100, A38524500 or Qubit Q32851, Q32854 |
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.
Step 2: Reverse transcribe RNA into cDNA
For best results, generate first strand cDNA using SuperScript IV Reverse Transcriptaseand generate second strand cDNA using RNaseOUT Recombinant Ribonuclease Inhibitor.
SuperScript IV Reverse Transcriptase user guide
RNaseOUT Recombinant Ribonuclease Inhibitor user guide
Step 3: Perform PCR enrichment
Follow recommendations for sequencing SARS-CoV-2 using Platinum SuperFi U Multiplex Master Mix.
Platinum SuperFi U Multiplex Master Mix user guide
Check the ARTIC Network website for the ARTIC primer pool latest release.
Note: We strongly recommend performing a gradient PCR to determine the optimal annealing temperature for your thermocycler. Subtle differences in thermocycler calibration can result in specific amplicons dropping out. Reducing our annealing temperature from 65°C to 63°C for identical cDNA input recovered amplicon.
Perform PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide. For further recommendations, refer to Step 4 below
Step 4: 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 | PCR mix cleanup | |||||
|---|---|---|---|---|---|---|---|
| 1. Remove EDTA from DNA samples | (if needed) | PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide | |||||
| Input: 1–500 ng | Input: 50 ng of combined amplicon pool | ||||||
| 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 | Combined amplicon pools (50 ng) | 40 µL | ||||
| Double-stranded DNA (1 ng - 500 ng) | X µL | 10X Fragmentation and dA-tailing Buffer (blue) | 5 µL | ||||
| 10X Fragmentation and dA-tailing Buffer (blue ) | 5 µL | Total volume (light blue mixture) | 45 µL | ||||
| Total volume (light blue mixture ) | 40 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 | ||||||
| Buffer-DNA mixture from step 1 (light blue mixture) | 40 µL | ||||||
| 5X Fragmentation and dA-tailing Enzyme Mix (clear) | 10 µL | ||||||
| Total volume (light blue mixture) | 50 µL | ||||||
| Recommended fragmentation time and optimization range to attain the desired fragment size | Fragment for 15 minutes at 37℃ Seal the plate and incubate the mixture in a thermal cycler with the heated lid set to 80–85°C, the block pre-cooled to 4°C, and programmed as outlined in the following table: | ||||||
| Fragment size | Fragmentation time at 37°C | Step | Temperature | Time | |||
| Recommen-dation | Recommen-dation | Pre-cool the block | 4ºC | As required | |||
| 150-300 bp | 20 min | 20-30 min | Fragmentation | 37ºC | 15 minutes | ||
| 300-500 bp | 10 min | 10-20 min | dA-tailing | 65ºC | 10 minutes | ||
| 500-700 bp | 5 min | 5-10 min | Hold | 4ºC | 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.
Note: qPCR is the recommended QC method for NGS due to the technological advantage to discriminate between adapter having and lacing library fragments. We recommend using Collibri Library Quantification Kit
Throughput | Recommendation |
|---|---|
120 preparations per run | For mid-throughput with full Collibri kits, use A38605024 (CD) with A38607196 (UD) together or any UD set of 24 preparations with A38607096 (CD). |
192 preparations per run | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD). |
| >192 preparations per run | For unlimited throughput request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W) |
Note: Do not pool together different sizes of kits containing the same type of indexed adaptors (CDs or UDs only). | |
Note: A38606024, A38605024, A43606024, and A43607024 (24 preparations) together are equivalent to A38607196 (96 preparations) and can be ordered interchangeably. | |
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.
Click image to enlarge
| 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 |
Click image to enlarge
| 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.
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, A38607196 |
| 4. Quantify libraries | Collibri Library Quantification Kit or Qubit fluorometric assays | qPCR A38524100, A38524500 or Qubit Q32850, Q32853 |
Procedure
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.
Step 2: Reverse transcribe RNA into cDNA
For best results, generate first strand cDNA using SuperScript IV Reverse Transcriptaseand generate second strand cDNA using Second Stand cDNA Synthesis Kit
SuperScript IV Reverse Transcriptase user guide
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.
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-HCl, 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 | ||
| 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 | ||||
| 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: (Suggested) 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 |
|
| 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 | 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.
With the continuous rise of new SARS-CoV-2 variants, there's a need for solid, precise, and quick sequencing strategies. The workflow instructions below are optimized for the study of coronaviruses following amplicon sequencing protocol originally created by ARTIC Network.
We currently support two protocols with PCR enrichment:
1. Recommended protocol: A workflow using SuperScript IV Vilo Master Mix and Phusion(TM) Plus Master Mix for cDNA synthesis and PCR enrichment, respectively.
2. Alternative protocol: A workflow using SuperScript IV RT and Platinum SuperFi U MasterMix for cDNA synthesis and PCR enrichment, respectively.
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
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 | 11756050, 11756500 |
| 3. Enrich PCR | ARTIC v3 primer pools Phusion Plus PCR Master Mix | Check the ARTIC Network website for the latest release. F631XL, F631L |
| 4. Prepare NGS libraries | Collibri ES DNA Library Prep Kit for Illumina Systems *The kit contains adapters with unique dual indexes (UDs) or combinatorial dual indexes (CDs). | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD) to support up to 192 preparations per run. See Table 7 for more recommendations. |
| 5. Quantify libraries | Collibri Library Quantification Kit or Qubit dsRNA HS Assay Kit | qPCR (recommended method) A38524100, A38524500 or Qubit Q32851, Q32854 |
Step 1: Perform RNA purification
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.
Step 2: Reverse transcribe RNA into cDNA
Generate cDNA using SuperScript IV VILO Master Mix according to the following recommendations:
Table 2. Recommendations for reaction set-up and cycling for cDNA generation.
| Component | Volume 10 µL rxn |
|---|---|
| Water, nuclease free | 2.5 µL |
| SuperScript IV VILO Master Mix | 2 µL |
| Purified RNA | 5.5 µL |
| Temperature | Time |
|---|---|
| 25ºC | 10 min |
| 50ºC | 10 min |
| 85ºC | 5 min |
Step 3: Perform PCR enrichment
Perform PCR enrichment (two reactions by sample) using the ARTIC primer pool according to the following recommendations.
Table 3. Reaction set-up for enriching viral sequences
| Component | Volume, 25 µL rxn |
|---|---|
| Water, nuclease free | 9 µL |
| 2X Phusion Plus PCR Master Mix | 12.5 µL |
| Primer Pool (1 or 2) | 1 µL |
| cDNA from previous reaction | 2.5 µL |
Check the ARTIC Network website for the ARTIC primer pool latest release.
Table 4. Cycling parameter for PCR enrichment
| Temperature | Time | Cycles |
|---|---|---|
| 98ºC | 30 sec | 1 |
| 98ºC | 15 sec | 30 |
| 63ºC | 5 min | |
| 4ºC | hold | 1 |
Combine 12.5 µL of each pool (25 µL in total) and perform PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide.
Step 4: Prepare NGS libraries
The following modifications are recommended to the Collibri ES DNA Library Prep Kit for Illumina Systems standard protocol.
- 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.
Table 5. Fragmentation and dA-tailing reactions - Incubate for 15 minutes at 37°C followed by 10 minutes at 65°C.
- Add 10 µL of the 7X Ligation mix and 10 µL of the adapter mix each sample on ice, and incubate for 30 minutes at 20ºC.
- Perform the post-ligation purification with magnetic beads from Collibri ES DNA Library Prep Kit Pub. No. MAN0019527.
- Amplify the library according to the following recommendations.
- Purify the amplified libraries with the magnetic beads from Collibri ES DNA Library Prep Kit, analyzed by Agilent 2100 Bioanalyzer.
| Component | Volume |
|---|---|
| Purified DNA | 18 µL |
| Elution Buffer | 17 µL |
| 10X Fragmentation and dA-tailing Buffer (blue) | 5 µL |
| 5X Fragmentation and dA-tailing Enzyme Mix | 10 µL |
| Total volume (light blue mixture) | 50 µL |
Table 6. Library amplification reaction set-up and cycling.
| Component | Volume, 25 µl rxn |
|---|---|
| Purified DNA | 20 µL |
| 2X Library Amplification Master Mix | 25 µL |
| Primer mix | 5 µL |
| Temperature | Time | Cycles |
|---|---|---|
| 98ºC | 30 sec | 1 |
| 98ºC | 15 sec | 3 |
| 60ºC | 30 sec | |
| 72ºC | 30 sec | |
| 72ºC | 60 sec | 1 |
| 4ºC | Hold | 1 |
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.
Note: qPCR is the recommended QC method for NGS due to the technological advantage to discriminate between adapter having and lacing library fragments. We recommend using Collibri Library Quantification Kit
| Throughput | Recommendationn |
|---|---|
| 120 preparations per run | For mid-throughput with full Collibri kits, use A38605024 (CD) with A38607196 (UD) together or any UD set of 24 preparations with A38607096 (CD). |
| 192 preparations per run | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD). |
| >192 preparations per run | For unlimited throughput request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W). |
| Note: Do not pool together different sizes of kits containing the same type of indexed adaptors (CDs or UDs only). | |
| Note: A38606024, A38605024, A43606024, and A43607024 (24 preparations) together are equivalent to A38607196 (96 preparations) and can be ordered interchangeably. | |
Alternative protocol
The optimized Collibri Next-Generation Sequencing of SARS-CoV-2 protocol using PCR enrichment has been used to sequence a total of 1001 SARS-CoV-2 positive clinical samples. As evident in Figure 1, a targeted amplicon-based sequencing approach is not only cost-effective, but also provides high-quality results. Libraries were sequenced on the Illumina MiSeq system by paired-end sequencing of 2 x 150 bp, including a 10% v/v PhiX sequencing control. Alignment was performed with BWA (bwa mem -t 32 -r 1.0 -k 19 -M -B 6 -v 1). The mean coverage, or the average number of reads that align to the reference NCBI ASM985889v3 genome, has been calculated by QualiMap BamQC.
Collection 1 | Collection 2 | Collection 3 | Collection 4 | Collection 5 | Collection 6 | |
|---|---|---|---|---|---|---|
Number of samples | 93 | 192 | 192 | 192 | 140 | 192 |
Mean aligned reads (%) | 97.3 | 70.2 | 85.9 | 85.6 | 91.0 | 86.4 |
Mean aligned reads (counts) | 134,623 | 26,335 | 91,653 | 60,920 | 121,623 | 91,653 |
Figure 1. Percentage of samples with different depth of coverage within each of six collections. A total of 1001 SARS-CoV-2 positive clinical samples were sequenced. The results show that amplicon-based workflow solution using Collibri ES DNA Library Prep Kit for Illumina Systems enables population-wide viral surveillance and research.
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 Reverse Transcriptase and RNaseOUT Recombinant Ribonuclease Inhibitor and dNTP Mix (10 mM each) and Random Hexamer Primer | 18090050 and 10777019 and 18427013 and SO142 |
| 3. Enrich PCR | ARTIC v3 primer pools Platinum SuperFi U Multiplex Master Mix | Check the ARTIC Network website for the latest release. A5140024 or A5140096 |
| 4. Prepare NGS libraries | Collibri ES DNA Library Prep Kit for Illumina Systems | Combinatorial Dual indexes (CD) A38605024 (24 preparations), Unique Dual Indexes (UD) A38606024, A43605024, A43606024 and A43607024 (24 preparations) or See Table 3 for more recommendations. |
| 5. Quantify libraries | Collibri Library Quantification Kit or Qubit dsRNA HS Assay Kit | qPCR A38524100, A38524500 or Qubit Q32851, Q32854 |
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.
Step 2: Reverse transcribe RNA into cDNA
For best results, generate first strand cDNA using SuperScript IV Reverse Transcriptaseand generate second strand cDNA using RNaseOUT Recombinant Ribonuclease Inhibitor.
SuperScript IV Reverse Transcriptase user guide
RNaseOUT Recombinant Ribonuclease Inhibitor user guide
Step 3: Perform PCR enrichment
Follow recommendations for sequencing SARS-CoV-2 using Platinum SuperFi U Multiplex Master Mix.
Platinum SuperFi U Multiplex Master Mix user guide
Check the ARTIC Network website for the ARTIC primer pool latest release.
Note: We strongly recommend performing a gradient PCR to determine the optimal annealing temperature for your thermocycler. Subtle differences in thermocycler calibration can result in specific amplicons dropping out. Reducing our annealing temperature from 65°C to 63°C for identical cDNA input recovered amplicon.
Perform PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide. For further recommendations, refer to Step 4 below
Step 4: 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 | PCR mix cleanup | |||||
|---|---|---|---|---|---|---|---|
| 1. Remove EDTA from DNA samples | (if needed) | PCR mix cleanup according to the Removal of EDTA from DNA samples section of the Collibri ES DNA Library Prep Kit for Illumina Systems user guide | |||||
| Input: 1–500 ng | Input: 50 ng of combined amplicon pool | ||||||
| 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 | Combined amplicon pools (50 ng) | 40 µL | ||||
| Double-stranded DNA (1 ng - 500 ng) | X µL | 10X Fragmentation and dA-tailing Buffer (blue) | 5 µL | ||||
| 10X Fragmentation and dA-tailing Buffer (blue ) | 5 µL | Total volume (light blue mixture) | 45 µL | ||||
| Total volume (light blue mixture ) | 40 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 | ||||||
| Buffer-DNA mixture from step 1 (light blue mixture) | 40 µL | ||||||
| 5X Fragmentation and dA-tailing Enzyme Mix (clear) | 10 µL | ||||||
| Total volume (light blue mixture) | 50 µL | ||||||
| Recommended fragmentation time and optimization range to attain the desired fragment size | Fragment for 15 minutes at 37℃ Seal the plate and incubate the mixture in a thermal cycler with the heated lid set to 80–85°C, the block pre-cooled to 4°C, and programmed as outlined in the following table: | ||||||
| Fragment size | Fragmentation time at 37°C | Step | Temperature | Time | |||
| Recommen-dation | Recommen-dation | Pre-cool the block | 4ºC | As required | |||
| 150-300 bp | 20 min | 20-30 min | Fragmentation | 37ºC | 15 minutes | ||
| 300-500 bp | 10 min | 10-20 min | dA-tailing | 65ºC | 10 minutes | ||
| 500-700 bp | 5 min | 5-10 min | Hold | 4ºC | 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.
Note: qPCR is the recommended QC method for NGS due to the technological advantage to discriminate between adapter having and lacing library fragments. We recommend using Collibri Library Quantification Kit
Throughput | Recommendation |
|---|---|
120 preparations per run | For mid-throughput with full Collibri kits, use A38605024 (CD) with A38607196 (UD) together or any UD set of 24 preparations with A38607096 (CD). |
192 preparations per run | For the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD). |
| >192 preparations per run | For unlimited throughput request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W) |
Note: Do not pool together different sizes of kits containing the same type of indexed adaptors (CDs or UDs only). | |
Note: A38606024, A38605024, A43606024, and A43607024 (24 preparations) together are equivalent to A38607196 (96 preparations) and can be ordered interchangeably. | |
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.
Click image to enlarge
| 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 |
Click image to enlarge
| 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.
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, A38607196 |
| 4. Quantify libraries | Collibri Library Quantification Kit or Qubit fluorometric assays | qPCR A38524100, A38524500 or Qubit Q32850, Q32853 |
Procedure
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.
Step 2: Reverse transcribe RNA into cDNA
For best results, generate first strand cDNA using SuperScript IV Reverse Transcriptaseand generate second strand cDNA using Second Stand cDNA Synthesis Kit
SuperScript IV Reverse Transcriptase user guide
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.
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-HCl, 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 | ||
| 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 | ||||
| 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: (Suggested) 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 |
|
| 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 | 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.
Resources
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Technical notes
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