Aug 24, 2022
Version 4
Enhanced QIAseq DIRECT SARS-CoV-2 Kit for Illumina MiSeq V.4
- 1FDA CFSAN
Protocol Citation: Padmini Ramachandran, Tamara Walsky, Amanda Windsor, Maria Hoffmann, Christopher Grim 2022. Enhanced QIAseq DIRECT SARS-CoV-2 Kit for Illumina MiSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy39rlx1/v4Version created by Padmini Ramachandran
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 24, 2022
Last Modified: August 24, 2022
Protocol Integer ID: 69118
Keywords: QIASeq Direct SARS COV-2, Miseq, SARSCOV-2 Enrichment
Abstract
- This SARS-CoV-2 targeted, a rapid viral RNA library preparation kit compatible with Illumina sequencers, provides an approximately five hour turn-around time from viral RNA sample to sequencing. The kit includes cDNA synthesis, SARS-CoV-2 enrichment, and library amplification and indexing reagents. The Enhanced protocol removes the need for normalization and quantification prior to amplification and indexing. RNA input is 5 μl, regardless of SC2 viral load in the sample extract. Amplification is performed using two primer pools that produce overlapping amplicons that are approximately 250 bp in size; as a result, no fragmentation is necessary prior to sequencing on Illumina platforms. 10 bp unique dual indexes (UDIs) are provided in the kit, and with a cycle read length of 149 for each paired-end read, the sequencing time is just over 28 hours.
- Version Updates: V3- Recommending the addition of the QIAseq DIRECT Booster Panel A Booster primer panel for QIAseq DIRECT SARS-CoV-2 to increase coverage across multiple regions of the SARS-CoV-2 genome.
- Note: This booster panel is designed to overcome S Gene Target Failure (SGTF).
Materials
Enhanced QIAseq DIRECT SARS-CoV-2 Kit
IMPORTANT: THIS PROTOCOL USES QIAseq DIRECT SARS-CoV-2 Enhancer. To use this protocol, you purchase QIAseq DIRECT SARS-CoV-2 Kit and QIAseq DIRECT SARS-CoV-2 Enhancer.
QIAseq DIRECT SARS-CoV-2 Kit Catalog no.
A (333891), B (333892),
C (333893), D (333894),
E (333886), F (333887),
G (333888), H (333889)
Note: You would need only one index kit with the QIAseq kit. For example if you need QIAseq DIRECT SARS-CoV-2 Kit with index kit A, you will place the order for catalog no: 333891
Components of A (333891), B (333892), C (333893), D (333894), E (333886), F (333887), G (333888), H (333889)
RP Primer, 12 μl 1 tube
EZ Reverse Transcriptase 1 tube
RNase Inhibitor 1 tube
Multimodal RT Buffer, 5x 1 tube
QIAseq 2X HiFi MM 4 tubes (not used in the Enhanced QIAseq DIRECT SARS-CoV-2 Kit protocol)
DIRECT SARS-CoV-2 Pool 1 1 tube
DIRECT SARS-CoV-2 Pool 2 1 tube
QIAseq DIRECT UDI Set Variable (A, B, C, D, E, F, G, or H)
Nuclease-Free Water 1 bottle
QIAseq Beads 1 bottle
QIAseq DIRECT Booster Panel A- Catalog No. -333897
QIAseq DIRECT SARS-CoV-2 Enhancer Catalog no. 333884
Components
RP Primer, 96 μl 1 tube
UPCR Buffer, 5x 2 tubes
QN Taq Polymerase 1 tube
Note: RP Primer, 12 µl (from QIAseq DIRECT SARS-CoV-2 Kit) and RP Primer, 96 µl (from QIAseq DIRECT SARS-CoV-2 Enhancer) are the same formulation and can be combined/used together.
cDNA Synthesis Procedure
cDNA Synthesis Procedure
Note
cDNA Synthesis Procedure: Important points before starting
- Use 5 μl viral RNA input, regardless of viral titer.
- Set up cDNA synthesis reaction on ice.
- Use a thermal cycler with a heated lid.
Thaw template RNA on ice. Gently yet thoroughly mix, then briefly centrifuge to collect residual liquid from the sides of the tubes and return to ice.
Note
Prepare the reagents required for cDNA synthesis.
- Thaw RP Primer (random hexamer), Multimodal RT Buffer, and nuclease-free water at room temperature (15–25°C).
- Mix thoroughly and then briefly centrifuge to collect residual liquid from the sides of the tubes. Prolonged storage of Multimodal RT Buffer at -20°C can cause white precipitate to form. This is normal. Just ensure to mix until precipitate is fully dissolved.
- Thaw RNase Inhibitor and EZ Reverse Transcriptase on ice. Mix by flicking the tubes, briefly centrifuge, and return to ice. After use, immediately return the enzymes to the freezer.
While keeping reagents on ice, prepare the cDNA reaction according to the table below. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again. For multiple reactions, prepare the calculated volume for all reactions, plus 10%.
| Components | Volume/reaction(ul) | |
| EZ Reverse Transcriptase | 1 | |
| Multimodal RT Buffer, 5x | 4 | |
| Nuclease-free water | 8 | |
| RNase Inhibitor | 1 | |
| RP Primer | 1 | |
| Template RNA | 5 | |
| Total volume | 20 |
cDNA synthesis incubation. Incubate as described below.
| A | B | C | |
| Step | Temperature (C) | Incubation time (min) | |
| 1 | 25 | 10 | |
| 2 | 42 | 50 | |
| 3 | 85 | 5 | |
| 4 | 4 | Hold |
Proceed to“ SARS-CoV-2 Enrichment Procedure”. Alternatively, the samples can be stored at−30 to–15°C in a constant-temperature freezer.
Note
SARS-CoV-2 Enrichment Procedure: Important points before starting.
- Use a thermal cycler with heated lid.
- Set up the reactions on ice.
- Vortex the QIAseq Beads thoroughly to ensure they are completely resuspended; the solution should be a homogenous brown color. If a delay in the protocol occurs, simply vortex the beads before using.
- Important: Prepare fresh 80% ethanol daily.
SARS-CoV-2 Enrichment Procedure
SARS-CoV-2 Enrichment Procedure
Note
Prepare the reagents required for target enrichment.
- Thaw the DIRECT SARS-CoV-2 Pool 1, DIRECT SARS-CoV-2 Pool 2, and 5x UPCR Buffer at room temperature. “Gently yet thoroughly” vortex to mix, and then centrifuge briefly.
- Thaw the QN Taq Polymerase on ice. Mix by flicking the tubes, and then centrifuge briefly.
While keeping reagents on ice, set up Pool 1 and Pool 2 reactions for each sample, as described below. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.
| A | B | C | |
| Component | Pool 1: Volume/reaction (µl) | Pool 2: Volume/reaction (µl) | |
| cDNA from “Protocol: Enhanced QIAseq DIRECT” | 8 | 8 | |
| DIRECT SARS-CoV-2 Pool 1 | 2 | --- | |
| DIRECT SARS-CoV-2 Pool 2 | --- | 2 | |
| DIRECT Pool 1 Booster A | 2 | 0 | |
| DIRECT Pool 2 Booster A | 0 | 2 | |
| UPCR Buffer, 5x | 5 | 5 | |
| QN Taq Polymerase | 1 | 1 | |
| Nuclease-free water | 7 | 7 | |
| Total volume | 25 | 25 |
Important: For samples with a broad/unknown range of Ct values (i.e., above and below Ct = 32) incubate as described in Table 9.1. For samples with Ct > 32, also incubate as described in Table 9.1. For samples with Ct value < 32, incubate as described in Table 9.2.
Target enrichment cycling: Samples with broad/unknown range Ct values and samples with Ct > 32
| A | B | C | D | |
| Step | Time | Temperature (°C) | Number of cycles | |
| Heat activation | 2 min | 98 | 1 | |
| Denaturation | 20 s | 98 | 4 | |
| Annealing/extension | 5 min | 63 | ||
| Denaturation | 20 s | 98 | 29 | |
| Annealing/extension | 3 min | 63 | ||
| Final Hold | ∞ | 4 | Hold |
Target enrichment cycling: Samples with Ct < 32
| A | B | C | D | |
| Step | Time | Temperature (°C) | Number of cycles | |
| Heat activation | 2 min | 98 | 1 | |
| Denaturation | 20 s | 98 | 4 | |
| Annealing/extension | 5 min | 63 | ||
| Denaturation | 20 s | 98 | 20 | |
| Annealing/extension | 3 min | 63 | ||
| Final Hold | ∞ | 4 | Hold |
After amplification, combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single well of a strip/plate, giving a volume of 50 µl.
Add 50 µl QIAseq Beads to each 50 µl combined sample. Briefly centrifuge ,“gently yet thoroughly” vortex to mix, and centrifuge briefly again.
Note
Note: Vortex the QIAseq Beads thoroughly to ensure they are completely resuspended; the solution should be a homogenous brown color.
Incubate for 5min at room temperature.
Centrifuge the strip until the beads are pelleted (2 min), and then place the plate onto a magnetic rack. After the solution has cleared (2 min or longer), carefully remove and discard the supernatant.
A- Top left: After pooling PCR rxns, add 50 µl room temp QIAseq Beads to each 50 µl rxn and perform bead clean up.
B: Top right: Example of beads beginning to pellet.
C: Bottom left: Not quite pelleted. NOTE: At this point, ~2 min should do it.
D. Fully pelleted beads. NOTE: Solution is completely clear.
Note
Important: Do not discard the beads, as they contain the DNA of interest.
Tip: It may be valuable to discard the supernatant twice. The contents settle after the first aspiration.
With the strip still on the magnetic stand, add 200 µl of 80% ethanol. Wait 1 min and carefully remove and discard the wash. Repeat the wash, for a total of 2 ethanol washes. Remove excess ethanol as much as possible.
Note
Important: To, completely remove all traces of the ethanol wash after the second wash, immediately briefly centrifuge and return the tubes to the magnetic stand. Remove the ethanol with a 200 μl pipette first, and then use a 10 μl pipette to remove any residual ethanol. This should be done quickly.
With the strip still on the magnetic stand, air-dry at room temperature for 4–8 min (until the beads start to crack and pellet loses its shine).
- Air dry the beads until ALL EtOH is removed and the beads appear cracked (see image)
- To check pellets, turn tube around and look at pellet from back. Holding tubes in front of a sheet of white paper increases ability to visualize cracks.
Remove the strip from the magnetic stand and elute the DNA from the beads by adding 30 μl nuclease-free water. “Gently yet thoroughly” vortex (triturate if necessary) to mix, briefly centrifuge, incubate for 2 min.
Centrifuge the strip until the beads are pelleted (2 min), and then return the plate to the magnetic rack until the solution has cleared.
Transfer 28 μl to a clean plate. This is now “enriched SARS-CoV-2”.
Assess the enriched SARS CoV-2 using Tape station HS D5000 tape. Also, quantify using Qubit HS kit.
Tape station image of cleaned enriched SARS- COV-2 amplicon from a high positive wastewater sample.
Important Notes:
- With wastewater samples anything that is 1ng/µl and above (even down to 0.5ng/µl with high Ct values) is a success and should definitely move on to final amplification!
Proceed to “Library Amplification and Indexing Procedure”. Alternatively, the samples can be stored at –30 to –15°C in a constant-temperature freezer.
Library Amplification and Indexing Procedure
Library Amplification and Indexing Procedure
Note
Library Amplification and Indexing Procedure: Important points before starting
- The QIAseq DIRECT UDI Sets have pierceable foil seals, and the indexes must be pipetted from the plate into separate reaction plates. To prevent cross-contamination, each well is single use.
- Set up the reactions on ice.
- Use a thermal cycler with a heated lid.
- Equilibrate the QIAseq Beads to room temperature (15–25°C) for 20–30 min before use.
- Vortex the QIAseq Beads thoroughly to ensure they are completely resuspended; the solution should be a homogenous brown color. If a delay in the protocol occurs, simply vortex the beads before using.
- Important: Prepare fresh 80% ethanol daily.
Note
Prepare the reagents required for library amplification and indexing.
- Thaw the QIAseq DIRECT UDI Index Set A, B, C, or D, as well as the UPCR Buffer, 5x, at room temperature. “Gently yet thoroughly” vortex to mix, and then centrifuge briefly.
- Dilute the index primer plate (1:10) dilution on a new 96 well plate. Use the diluted indices for step 25. Save the remaining diluted indices at -20C for future use.
- Thaw the QN Taq Polymerase. Mix by flicking the tubes, and then centrifuge briefly.
For samples with a broad/unknown range of Ct values (i.e., above and below Ct = 32) incubate as described in Table 23.1. For samples with Ct > 32, also incubate as described in Table 23.1. For samples with Ct value < 32, incubate as described in Table 23.2.
Library amplification/indexing cycling conditions: Samples with broad/unknown range Ct values and samples with Ct > 32
| A | B | C | D | |
| Step | Temperature (°C) | Time | Number of cycles | |
| Initial denaturation | 98 | 2 min | 1 | |
| 3-step cycling | ||||
| Denaturation | 98 | 20 s | ||
| Annealing | 60 | 30 s | 11 | |
| Extension | 72 | 30 s | ||
| Final extension | 72 | 1 min | 1 | |
| Final Hold | 4 | ∞ | Hold |
Library amplification/indexing cycling conditions: Samples with Ct < 32
| A | B | C | D | |
| Step | Temperature (°C) | Time | Number of cycles | |
| Initial denaturation | 98 | 2 min | 1 | |
| 3-step cycling | ||||
| Denaturation | 98 | 20 s | ||
| Annealing | 60 | 30 s | 14 | |
| Extension | 72 | 30 s | ||
| Final extension | 72 | 1 min | 1 | |
| Hold | 4 | ∞ | Hold |
On ice, prepare the library amplification and indexing reaction according to Table in Step 25. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.
Note
Note: If setting up more than one reaction, prepare a volume of master mix 10% greater than what is required for the total number of reactions.
Reaction mix for library amplification and indexing
| A | B | |
| Component | Volume/reaction | |
| “Enriched SARS-CoV-2” sample | 24 μl | |
| Index from QIAseq DIRECT UDI index plate (A, B, C, or D) Plate, 1:10 dilution | 2 μl | |
| Nuclease-free water | 12 μl | |
| UPCR Buffer 5x | 10 μl | |
| QN Taq Polymerase | 2 μl | |
| Total reaction volume | 50 μl |
Transfer the strip to the thermal cycler and start the program.
Once PCR is complete, add 40 μl of resuspended QIAseq Beads to each 50 μl reaction. Briefly centrifuge, “gently yet thoroughly” vortex to mix, and centrifuge briefly again.
Note
Note: Vortex the QIAseq Beads thoroughly to ensure they are completely resuspended; the solution should be a homogenous brown color.
Incubate the mixture for 5 min at room temperature.
Centrifuge the strip until the beads are pelleted (2 min), and then place the strip onto a magnetic rack. After the solution has cleared (2 min or longer), carefully remove and discard the supernatant.
Note
Important: Do not discard the beads as they contain the DNA of interest.
Tip: It may be valuable to discard the supernatant twice. The contents settle after the first discard.
With the strip still on the magnetic stand, add 200 μl of 80% ethanol. Wait 1 min and carefully remove and discard the wash. Repeat the wash, for a total of 2 ethanol washes. Remove excess ethanol as much as possible.
Note
Important: To completely remove all traces of the ethanol wash after the second wash, immediately briefly centrifuge and return the strip to the magnetic stand. Remove the ethanol with a 200 μl pipette first, and then use a 10 μl pipette to remove any residual ethanol. This should be done quickly.
With the strip still on the magnetic stand, air-dry at room temperature for 4–8 min (until the beads start to crack and pellet loses its shine).
- Air dry the beads until ALL EtOH is removed and the beads appear cracked (see image).
- To check pellets, turn tube around and look at pellet from back. Holding tubes in front of a sheet of white paper increases ability to visualize cracks.
Remove the strip from the magnetic stand and elute the DNA from the beads by adding 25 μl nuclease-free water. Vigorously vortex (triturate if necessary) to mix, briefly centrifuge, and incubate for 2 min.
Centrifuge the strip until the beads are pelleted (2 min), and then return the plate to the magnetic rack until the solution has cleared (2 min or longer).
Transfer 23 μl to a clean strip. This is the “SARS-CoV-2 library”. If not proceeding immediately, the sample can be stored at –30 to –15°C.
Quantify the library using Qubit HS assay kit.
Assess the quality of the library using a Bioanalyzer, Tape Station (D1000 tape), or Fragment Analyzer. Check for the expected size distribution of library fragments.
Tape station image of Qiaseq library from high positive wastewater sample.
Quantification, Normalization and Pooling of Libraries
Quantification, Normalization and Pooling of Libraries
Using the attached Post library worksheet, calculate the nM based on the Qubit concentration (ng/ul) and the Tape station (bp) profile. Normalize the “SARS-CoV-2 library”, to 4 nM. Quantify the final pool at the end.
Postlibraryworksheet.xlsx Proceed to loading on to MiSeq (150x 150 cycle) using the attached Sample Sheet. Alternatively, the purified “SARS-CoV-2 library” can be safely stored at –30 to –15°C in a constant-temperature freezer until ready to use for sequencing. Controlled vocabulary for labelling/naming positiveControl and negativeControl has been added to the Sample sheet. SampleSheet.csv
SampleSheet.csv