NGS for COVID-19

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.

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.

Recommended protocol

 Download app note

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 RNAMagMAX Viral/Pathogen Nucleic Acid Isolation KitA48310A42352
2. Reverse transcribe RNA into cDNASuperScript IV VILO Master Mix11756050, 11756500
3. Enrich PCRARTIC v3 primer pools

Phusion Plus PCR Master Mix
Check the ARTIC Network website for the latest release.

F631XL, F631L
4. Prepare NGS librariesCollibri 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.

For unlimited throughput, request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W).

See Table 7 for more recommendations.

5. Quantify librariesCollibri Library Quantification Kit
or
Qubit dsRNA HS Assay Kit
qPCR (recommended method) A38524100A38524500
or
Qubit Q32851Q32854

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.

 Download app note

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 free2.5 µL
SuperScript IV VILO Master Mix2 µL
Purified RNA5.5 µL
TemperatureTime
25ºC10 min
50ºC10 min
85ºC5 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

ComponentVolume, 25 µL rxn
Water, nuclease free9 µL
2X Phusion Plus PCR Master Mix12.5 µL
Primer Pool (1 or 2)1 µL
cDNA from previous reaction2.5 µL

Check the ARTIC Network website for the ARTIC primer pool latest release.

Table 4. Cycling parameter for PCR enrichment

TemperatureTimeCycles
98ºC30 sec1
98ºC15 sec30
63ºC5 min
4ºChold1

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.

 Download user guide

  1. 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

  2. ComponentVolume
    Purified DNA18 µL
    Elution Buffer17 µL
    10X Fragmentation and dA-tailing Buffer (blue)5 µL
    5X Fragmentation and dA-tailing Enzyme Mix10 µL
    Total volume (light blue mixture)50 µL
  3. Incubate for 15 minutes at 37°C followed by 10 minutes at 65°C.
  4. 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.
  5. Perform the post-ligation purification with magnetic beads from Collibri ES DNA Library Prep Kit Pub. No. MAN0019527.
  6. Amplify the library according to the following recommendations.

  7. Table 6. Library amplification reaction set-up and cycling.

    ComponentVolume, 25 µl rxn
      Purified DNA20 µL
    2X Library Amplification Master Mix25 µL
    Primer mix5 µL
    TemperatureTimeCycles
    98ºC30 sec1
    98ºC15 sec

    3
    60ºC30 sec
    72ºC30 sec
    72ºC60 sec1
    4ºCHold1
  8. Purify the amplified libraries with the magnetic beads from Collibri ES DNA Library Prep Kit, analyzed by Agilent 2100 Bioanalyzer.


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

 Download user guide

Table 7. Recommendations for the throughput and alternative combinations:

ThroughputRecommendationn
120 preparations per runFor 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 runFor the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD).
>192 preparations per runFor 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.

Mean coverages across different sets of clinical samples
 

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.

StepKitCatalog numbers
1. Purify Total RNAMagMAX Viral/Pathogen Nucleic Acid Isolation KitA48310, A42352
2. Reverse transcribe RNA into cDNASuperScript 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 PCRARTIC v3 primer pools

Platinum SuperFi U Multiplex Master Mix
Check the ARTIC Network website for the latest release.
A5140024 or A5140096
4. Prepare NGS librariesCollibri ES DNA Library Prep Kit for Illumina Systems

Combinatorial Dual indexes (CD) A38605024 (24 preparations),
A38607096 (96 preparations)

Unique Dual Indexes (UD) A38606024, A43605024, A43606024 and A43607024 (24 preparations) or
A38607196 (96 preparations)

See Table 3 for more recommendations.

5. Quantify librariesCollibri Library Quantification Kit
or
Qubit dsRNA HS Assay Kit
qPCR A38524100, A38524500
or
Qubit Q32851Q32854


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


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.

 Download user guide

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

StepStandard recommendationPCR 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 ngInput: 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.   
ComponentVolume ComponentVolume
10 mM Tris-HCl, ph 7.5-8.5to 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 sizeFragmentation time at 37°CStep Temperature Time
Recommen-dationRecommen-dationPre-cool the block4ºCAs required
150-300 bp20 min20-30 minFragmentation37ºC15 minutes
300-500 bp10 min10-20 mindA-tailing65ºC10 minutes
500-700 bp5 min5-10 minHold4ºCHold


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.

 Download user guide

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

Table 3. Recommendations for the throughput and alternative combinations:

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 runFor 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.

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.

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.

 Download app note ›


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.

 Download user guide

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

StepStandard recommendationRecommended 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 ngInput: 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.   
ComponentVolumeComponentVolume
10 mM Tris-HCl, ph 7.5-8.5to 40 µLcDNA10 µL
Double-stranded DNA (1 ng - 500 ng)X µL10 mM Tris-HCl, pH 7.5-8.5to 31 µL
10X Fragmentation and dA-tailing Buffer (blue )5 µL10X Fragmentation and dA-tailing Buffer (blue)5 µL
Total volume (light blue mixture )40 µLTotal 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.
ComponentVolumeComponentVolume
Buffer-DNA mixture from step 1 (light blue mixture)40 µLBuffer-DNA mixture from step 1 (light blue mixture)36 µL
5X Fragmentation and dA-tailing Enzyme Mix (clear)10 µL5X Fragmentation and dA-tailing Enzyme Mix (clear)14 µL
Total volume (light blue mixture)50 µLTotal volume (light blue mixture)50 µL
 Recommended fragmentation time and optimization range to attain the desired fragment sizeFragment for 20 minutes at 37℃
Fragment sizeFragmentation time at 37°C
Recommen-dationOptimization range
150-300 bp20 min20-30 min
300-500 bp10 min10-20 min
500-700 bp5 min5-10 min
3. Carry out post-ligation double-sided size selectionDouble-sided size selection to match target insert sizeCustomized cleanup protocol
4. PCR amplify the libraryThe 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 goalRecommendationBenefits
SARS-CoV-2 genomic evolutionFollowing library prep, enrich for SARS-CoV-2
  • Enhanced percentage of reads mapping to SARS-CoV-2 
  • Reduced cost of sequencing
Host-pathogen interactionsNo 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.

StageNumber of cyclesTemperatureTime
Activate the enzyme1 cycle98°C30 seconds
Denature3-4 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°C15 seconds
Anneal60°C30 seconds
Extend72°C30 seconds
Final extension1 cycle72°C1 minutes
Hold1 cycle4°CHold


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.

 Download user guide

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.

Recommended protocol

 Download app note

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 RNAMagMAX Viral/Pathogen Nucleic Acid Isolation KitA48310A42352
2. Reverse transcribe RNA into cDNASuperScript IV VILO Master Mix11756050, 11756500
3. Enrich PCRARTIC v3 primer pools

Phusion Plus PCR Master Mix
Check the ARTIC Network website for the latest release.

F631XL, F631L
4. Prepare NGS librariesCollibri 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.

For unlimited throughput, request Collibri ES DNA Library Prep Core Kit without indexes (A38607096W).

See Table 7 for more recommendations.

5. Quantify librariesCollibri Library Quantification Kit
or
Qubit dsRNA HS Assay Kit
qPCR (recommended method) A38524100A38524500
or
Qubit Q32851Q32854

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.

 Download app note

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 free2.5 µL
SuperScript IV VILO Master Mix2 µL
Purified RNA5.5 µL
TemperatureTime
25ºC10 min
50ºC10 min
85ºC5 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

ComponentVolume, 25 µL rxn
Water, nuclease free9 µL
2X Phusion Plus PCR Master Mix12.5 µL
Primer Pool (1 or 2)1 µL
cDNA from previous reaction2.5 µL

Check the ARTIC Network website for the ARTIC primer pool latest release.

Table 4. Cycling parameter for PCR enrichment

TemperatureTimeCycles
98ºC30 sec1
98ºC15 sec30
63ºC5 min
4ºChold1

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.

 Download user guide

  1. 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

  2. ComponentVolume
    Purified DNA18 µL
    Elution Buffer17 µL
    10X Fragmentation and dA-tailing Buffer (blue)5 µL
    5X Fragmentation and dA-tailing Enzyme Mix10 µL
    Total volume (light blue mixture)50 µL
  3. Incubate for 15 minutes at 37°C followed by 10 minutes at 65°C.
  4. 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.
  5. Perform the post-ligation purification with magnetic beads from Collibri ES DNA Library Prep Kit Pub. No. MAN0019527.
  6. Amplify the library according to the following recommendations.

  7. Table 6. Library amplification reaction set-up and cycling.

    ComponentVolume, 25 µl rxn
      Purified DNA20 µL
    2X Library Amplification Master Mix25 µL
    Primer mix5 µL
    TemperatureTimeCycles
    98ºC30 sec1
    98ºC15 sec

    3
    60ºC30 sec
    72ºC30 sec
    72ºC60 sec1
    4ºCHold1
  8. Purify the amplified libraries with the magnetic beads from Collibri ES DNA Library Prep Kit, analyzed by Agilent 2100 Bioanalyzer.


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

 Download user guide

Table 7. Recommendations for the throughput and alternative combinations:

ThroughputRecommendationn
120 preparations per runFor 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 runFor the highest throughput with full Collibri kits, use A38607096 (CD) with A38607196 (UD).
>192 preparations per runFor 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.

Mean coverages across different sets of clinical samples
 

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.

StepKitCatalog numbers
1. Purify Total RNAMagMAX Viral/Pathogen Nucleic Acid Isolation KitA48310, A42352
2. Reverse transcribe RNA into cDNASuperScript 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 PCRARTIC v3 primer pools

Platinum SuperFi U Multiplex Master Mix
Check the ARTIC Network website for the latest release.
A5140024 or A5140096
4. Prepare NGS librariesCollibri ES DNA Library Prep Kit for Illumina Systems

Combinatorial Dual indexes (CD) A38605024 (24 preparations),
A38607096 (96 preparations)

Unique Dual Indexes (UD) A38606024, A43605024, A43606024 and A43607024 (24 preparations) or
A38607196 (96 preparations)

See Table 3 for more recommendations.

5. Quantify librariesCollibri Library Quantification Kit
or
Qubit dsRNA HS Assay Kit
qPCR A38524100, A38524500
or
Qubit Q32851Q32854


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


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.

 Download user guide

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

StepStandard recommendationPCR 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 ngInput: 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.   
ComponentVolume ComponentVolume
10 mM Tris-HCl, ph 7.5-8.5to 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 sizeFragmentation time at 37°CStep Temperature Time
Recommen-dationRecommen-dationPre-cool the block4ºCAs required
150-300 bp20 min20-30 minFragmentation37ºC15 minutes
300-500 bp10 min10-20 mindA-tailing65ºC10 minutes
500-700 bp5 min5-10 minHold4ºCHold


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.

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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

Table 3. Recommendations for the throughput and alternative combinations:

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 runFor 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.

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.

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.

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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.

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

StepStandard recommendationRecommended 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 ngInput: 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.   
ComponentVolumeComponentVolume
10 mM Tris-HCl, ph 7.5-8.5to 40 µLcDNA10 µL
Double-stranded DNA (1 ng - 500 ng)X µL10 mM Tris-HCl, pH 7.5-8.5to 31 µL
10X Fragmentation and dA-tailing Buffer (blue )5 µL10X Fragmentation and dA-tailing Buffer (blue)5 µL
Total volume (light blue mixture )40 µLTotal 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.
ComponentVolumeComponentVolume
Buffer-DNA mixture from step 1 (light blue mixture)40 µLBuffer-DNA mixture from step 1 (light blue mixture)36 µL
5X Fragmentation and dA-tailing Enzyme Mix (clear)10 µL5X Fragmentation and dA-tailing Enzyme Mix (clear)14 µL
Total volume (light blue mixture)50 µLTotal volume (light blue mixture)50 µL
 Recommended fragmentation time and optimization range to attain the desired fragment sizeFragment for 20 minutes at 37℃
Fragment sizeFragmentation time at 37°C
Recommen-dationOptimization range
150-300 bp20 min20-30 min
300-500 bp10 min10-20 min
500-700 bp5 min5-10 min
3. Carry out post-ligation double-sided size selectionDouble-sided size selection to match target insert sizeCustomized cleanup protocol
4. PCR amplify the libraryThe 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 goalRecommendationBenefits
SARS-CoV-2 genomic evolutionFollowing library prep, enrich for SARS-CoV-2
  • Enhanced percentage of reads mapping to SARS-CoV-2 
  • Reduced cost of sequencing
Host-pathogen interactionsNo 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.

StageNumber of cyclesTemperatureTime
Activate the enzyme1 cycle98°C30 seconds
Denature3-4 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°C15 seconds
Anneal60°C30 seconds
Extend72°C30 seconds
Final extension1 cycle72°C1 minutes
Hold1 cycle4°CHold


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.

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