Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples

Padmini Ramachandran, Tamara Walsky, Amanda Windsor, Maria Hoffmann, Christopher Grim

Published: 2022-03-10 DOI: 10.17504/protocols.io.b545q8y6

Disclaimer

This method is under development and assessment for suitability of use. It is likely that modifications will be made to improve the method.

Abstract

PURPOSE:

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI.

This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2. The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications. Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers. These primers are available in the NEBNext^®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina^®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep.

There are a couple of decision points in this protocol. Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions.

Version Updates: V2- Quality checks of cleaned amplicons on low viral titer and high viral titer samples has been added as a decision point before proceeding to sequencing.

Before start

Note: The amount of RNA required for detection depends on the abundance of the RNA of interest. In general, we recommend, using ≥ 10 copies of the (SARS-CoV-2) viral genome as input, however, results may vary depending on the quality of the input. In addition, we recommend setting up a no template control reaction and that reactions are set-up in a hood .

The presence of carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.

Steps

cDNA Synthesis

Note

The presence of genomic DNA or carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.We have also verified cDNA synthesis using the Invitrogen™ SuperScript™ IV First-Strand Synthesis System (Catalog number:18091200), as described in the SNAP protocol with modifications (random hexamers, RT incubation of 30 min.).

Gently mix and spin down the LunaScript RT SuperMix reagent. Prepare the cDNA synthesis reaction as described below:

A	B
COMPONENT	VOLUME
RNA Sample	8 µl
(lilac) LunaScript RT SuperMix	2 µl
Total Volume	10 µl

For no template controls, mix the following components:

A	B
COMPONENT	VOLUME
(white) Nuclease-free Water	8 µl
(lilac) LunaScript RT SuperMix	2 µl
Total Volume	10 µl

Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.

Incubate reactions in a thermocycler* with the following steps:

A	B	C	D
CYCLE STEP	TEMP	TIME	CYCLES
Primer Annealing	25°C	2 minutes	1
cDNA Synthesis	55°C	20 minutes
Heat Inactivation	95°C	1 minute
Hold	4°C	∞

*Set heated lid to 105°C

Note

Samples can be stored at for up to a week.

Targeted cDNA Amplification

Note

4.5 µl cDNA input is recommended. If using less than 4.5 µl of cDNA, add nuclease-free water to a final volume of 4.5 µl. We recommend setting up the cDNA synthesis and cDNA amplification reactions in different rooms to minimize cross-contamination of future reactions. The cDNA reaction can be set up in hood to minimize cross-contamination.

Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:

For Pool Set A:

A	B
COMPONENT	VOLUME
cDNA (Step 2)	4.5 µl
(lilac) Q5 Hot Start High-Fidelity 2X Master Mix	6.25 µl
NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1	1.75 µl
Total Volume	12.5 µl

For Pool Set B:

A	B
COMPONENT	VOLUME
cDNA (Step 2)	4.5 µl
(lilac) Q5 Hot Start High-Fidelity 2X Master Mix	6.25 µl
NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2	1.75 µl
Total Volume	12.5 µl

Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.

Incubate reactions in a thermocycler* with the following steps:

A	B	C	D
CYCLE STEP	TEMP	TIME	CYCLES
Initial Denaturation	98°C	30 seconds	1
Denature	95°C	15 seconds	35
Annealing/Extension	63°C	5 minutes
Hold	4°C	∞	1

*Set heated lid to 105°C

Combine the Pool A and Pool B PCR reactions for each sample.

Note

Samples can be stored at for up to a week.

10.

Assess the quantity of the cDNA amplicon using the Qubit HS kit. (1µL

11.

Assess the size of the cDNA amplicon using Agilent TapeStation System, Bioanalyzer, or similar size fragment analyzer.

TapeStation profile of cDNA amplicons of positive wastewater sample

TapeStation profile of cDNA amplicons of high Ct value or degraded wastewater sample. Notice measurable peak at 64 bp and unmeasurable peaks to the right.

TapeStation profile of cDNA amplicons of high Ct value or degraded wastewater sample. Notice unmeasurable peaks at approximately 250 and 450 bp.

TapeStation profile of cDNA amplicons of Twist SARS-CoV-2 control

12.

Note

Based on the viral load in the sample, you will see a spectrum of fragment profiles. If there is a single peak around 400-600 bp, the clean-up step can be omitted and you can proceed directly to library preparation using Illumina DNA Prep https://www.protocols.io/edit/modified-illumina-dna-prep-m-tagmentation-library-b2yzqfx6If there is not a single peak, check the amplicon size distributions. If there are lots of smaller peaks, for example, we typically see one at 44, 75, 128, and 213, these PCR reaction pools need to be cleaned up. Follow the cleanup protocol and prepare libraries using the Ultra II DNA workflow by NEB Next ARTIC SARS-CoV-2 Library Prep Kit (Illumina). If you are not able to perform the PCR pool assessment above, proceed to PCR cleanup and prepare libraries using the Ultra II DNA workflow by NEB Next ARTIC SARS-CoV-2 Library Prep Kit (Illumina).

Cleanup of cDNA Amplicons.

13.

Note

The volume of NEBNext Sample Purification Beads provided here are for use with the sample composition at this step (25 µl; Step 6). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.

14.

Vortex the NEBNext Sample Purification Beads to resuspend.

15.

Add 20µL to the combined PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

16.

Incubate samples at -20Room temperature for at least 0h 5m 0s.

17.

Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.

18.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

Note

Caution: do not discard the beads .

19.

Add 200µL to the tube/plate while in the magnetic stand. Incubate at -20Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

20.

Perform second Ethanol wash:

Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

21.

Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note

Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

22.

Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 28µL. If not assessing cDNA (Step 19) elute DNA in 27µL.

23.

Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at -20Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

24.

Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 26µL to a new PCR tube. If not assessing cDNA (Step 19) transfer 25µL to a new PCR tube.

25.

We recommend assessing cDNA amplicon (from Step24) concentrations with a Qubit fluorometer.

Note

Amplicons may also be run on a Bioanalyzer or TapeStation® to confirm 400 bp size of amplicons. To run on a Bioanalyzer, dilute amplicon 10-fold with 0.1X TE Buffer and run on a DNA High Sensitivity ScreenTape. (See Figure 19 below for example of amplicon size profile on a Tapestation).

Figure 19: Example of cDNA amplicons generated from 1000 genome copies of SARS CoV-2 in the absence (A) and presence (B) of human primer controls.

Note

Samples can be stored at for up to a week.

Quality checks on cleaned amplicons before proceeding to library prep

26.

We have observed the following output for VSS amplicons based on viral titer in the wastewater samples:

1)Moderate to high viral titer samples: The cleaned amplicon quantity is expected to be 0.5 ng/µl to 8 ng/µl

2)Low viral titer samples: The cleaned amplicon quantity is expected to be 0 - 0.2 ng/µl

We recommend to proceed to library preparation for samples at 0.2 ng/µl or higher. Cleaned VSS amplicons below this value have not yielded sequencing results.

27.

We recommend assessing cDNA amplicon (from Step 23) on Tape Station or Bioanalyzer using High Sensitivity kit.

Note

Figure 20: Tape station profile of cleaned amplicon from a moderate to high positive sample. Figure 21: Tape station profile of cleaned amplicon from a low positive sample. Figure 22: Tape station profile of cleaned amplicon from a low positive sample measured at ~0 ng/ul.

NEBNext End Prep

28.

This is library preparation Option 1, and the most common that will be used. You will use the Ultra II DNA workflow as described in the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) (cat# E7650S/L), with NEBNext^®Multiplex Oligos for Illumina^®(96 Unique Dual Index Primer Pairs) (cat# E6440S/L).

29.

Add the following components to a sterile nuclease-free tube:

A	B
COMPONENT	VOLUME
(green) NEBNext Ultra II End Prep Enzyme Mix	1.5 µl
(green) NEBNext Ultra II End Prep Reaction Buffer	3.5 µl
Targeted cDNA Amplicons*	25 µl
Total Volume	30 µl

*When cleanup of the pooled cDNA amplicons is skipped, the amplicons must be diluted prior to library prep, please dilute the cDNA as mentioned here. Transfer 2.5 µl of the pooled cDNA amplicons to a fresh tube. Add 22.5 µl of 0.1X TE for a final volume of 25 µl. If cleanup is performed, use the cleaned targeted cDNA amplicons as the input.

30.

Set a 100 µl or 200 µl pipette to 25 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

Note

It is important to mix well. The presence of a small amount of bubbles will not interfere with performance

31.

Place in a thermocycler* and run the following program:

A	B
TEMP	TIME
20°C	30 minutes
65°C	30 minutes
4°C	∞

*Set heated lid to 75°C

Note

If necessary, samples can be stored at ; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.

Adaptor Ligation

32.

Add the following components directly to the End Prep Reaction Mixture:

A	B
COMPONENT	VOLUME
End Prep Reaction Mixture (previous step)	30 µl
(red) NEBNext Adaptor for Illumina**	1.25 µl
(red) NEBNext Ultra II Ligation Master Mix*	15 µl
Total Volume	46.25 µl

Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.** The NEBNext adaptor is provided in NEBNext Oligo kits. NEB has several oligo options which are supplied separately from the library prep kit. Please see www.neb.com/oligos for additional information.

Note

Do not premix adaptor with the Ligation Master Mix.

33.

Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

Note

Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.

34.

Incubate at 20°C for 0h 15m 0s in a thermocycler with the heated lid off.

35.

Add 1.5µL to the ligation mixture from the previous step.

Note

Steps 26 and 27 are only required for use with NEBNext non-indexed Adaptors. USER enzyme can be found in the NEBNext Multiplex Oligos (www.neb.com/oligos).

36.

Mix well and incubate at 37°C for 0h 15m 0s with the heated lid set to ≥ 47°C.

Note

Samples can be stored overnight at .

Cleanup of Adaptor-ligated DNA

37.

The following section is for cleanup of the ligation reaction.

Note

The volume of NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step (47.75 µl; Step 27). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.

38.

Vortex the NEBNext Sample Purification Beads to resuspend.

39.

Add 43µL to the Adaptor Ligation reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

40.

Incubate samples on bench top for at least 0h 5m 0s at 47Room temperature.

41.

Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.

42.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

Note

Caution: do not discard beads .

43.

Add 200µL to the tube/ plate while in the magnetic stand. Incubate at 47Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

44.

Repeat the previous step once for a total of two washes:

Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

45.

Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

46.

Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 10µL.

47.

Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at 47Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

48.

Place the tube/plate on the magnetic stand. After 0h 5m 0s (or when the solution is clear), transfer 7.5µL to a new PCR tube.

Note

Samples can be stored at .

PCR Enrichment of Adaptor-ligated DNA

49.

Note

We only recommend using NEBNext Oligo kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied ay 10 µM (combined). We observed addtion of spurious nucleotides when using primers supplied in separate tubes

50.

Add the following components to a sterile strip tube:

A	B
COMPONENT	VOLUME
Adaptor Ligated DNA Fragments (Step 45)	7.5 μl
(blue) NEBNext Library PCR Master Mix	12.5 μl
Index Primer Mix (E6440S)*	5.0 μl
Total Volume	25 μl

NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.

51.

Set a 100 μl pipette to 20 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.

52.

Place the tube on a thermocycler** and perform PCR amplification using the following PCR cycling conditions:

A	B	C	D
CYCLE STEP	TEMP	TIME	CYCLES
Initial Denaturation	98°C	30 seconds	1
Denaturation	98°C	10 seconds	6-8*
Annealing/Extension	65°C	75 seconds
Final Extension	65°C	5 minutes	1
Hold	4°C	∞

The number of PCR cycles recommended should be viewed as a starting point and may need to be optimized for particular sample types. If you have cleaned up cDNA as input, use 8 cycles. If you have diluted cDNA as input use 6/7 cycles.**Set heated lid to 105°C.

Cleanup of PCR Reaction

53.

Note

The NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.

54.

Vortex NEBNext Sample Purification Beads to resuspend.

55.

Add 22.5µL to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

56.

Incubate samples on bench top for at least 0h 5m 0s at Room temperature.

57.

Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.

58.

After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

Note

Caution: do not discard the beads.

59.

Add 200µL to the tube/plate while in the magnetic stand. Incubate at Room temperature for 0h 0m 30s, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.

60.

Repeat the previous step once for a total of two washes:

Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

61.

Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note

62.

Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17µL.

63.

Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 0h 2m 0s at Room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.

64.

Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15µL to a new PCR tube and store at -20°C.

65.

Check the size distribution on an Agilent Bioanalyzer or TapeStation. The sample may need to be diluted before loading. A peak size of ~520 bp is expected (Figure 56).

Note

Samples can be stored at or proceed to MiSeq denature and dilution guideline to load it on to the MiSeq.

Figure 56: Example of final library size distributions on a TapeStation. ARTIC SARS-CoV-2 librarieswere generated from 1000 viral copies in the absence (A) or presence (B) of the human primer controls.

66.

Sample Sheet generation for NEBNext^® ARTIC SARS-CoV-2 Library Prep Kit (Illumina^®) with E6440 Index plate.E6440_ Forward Strand Workflow Sample Sheet.csv

Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples

Disclaimer

Abstract

Before start

Steps

cDNA Synthesis

Targeted cDNA Amplification

Cleanup of cDNA Amplicons.

Quality checks on cleaned amplicons before proceeding to library prep

NEBNext End Prep

Adaptor Ligation

Cleanup of Adaptor-ligated DNA

PCR Enrichment of Adaptor-ligated DNA

Cleanup of PCR Reaction

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