Enhanced QIAseq DIRECT SARS-CoV-2 Kit for Illumina MiSeq

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.

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%.

A	B
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 freeze.

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
UPCR Buffer, 5x	5	5
QN Taq Polymerase	1	1
Nuclease-free water	9	9
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 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.

Incubate for 5min at room temperature.

Centrifuge the plate 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.

With the plate 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.

With the plate 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).

Remove the plate 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 plate 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”.

Important Notes:

• We are adding tails ONLY to SARS-CoV-2 sequence in the subsequent Library Amplification and Indexing Procedure, and that will keep all the contaminants at bay when you perform your final amplification.

• So, after you transferred the 28 µl of supernatant from your elution, this is a good time to get a feel for what you have if you want to do a mid-protocol check.

• Use a high sensitivity Qubit (for example) and check the concentration of the enrichment reactions after bead cleanup.

• 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.

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.

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	2 μl
Nuclease-free water	12 μl
UPCR Buffer 5x	10 μl
QN Taq Polymerase	2 μl
Total reaction volume	50 μl

Transfer the plate 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.

Incubate the mixture for 5 min at room temperature.

Centrifuge the plate 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.

With the plate 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.

With the plate 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).

Remove the plate 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 plate 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 plate. 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, TapeStation (D1000 tape), or Fragment Analyzer. Check for the expected size distribution of library fragments. An example library is shown here.

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.SampleSheet.csv