NEXTFLEX...13. Place the 96 well PCR plate on the magnetic stand at room temperature for 2 minutes or until the supernatant appears completely clear. 14. Do not discard the sample

KIT CONTAINS: 96 or 384 RXNSCompatible with Illumina® platforms

NEXTFLEX®

Variant-Seq™

SARS-CoV-2 Kit (For Illumina® Platforms)

APPLIED GENOMICS

v21.03

USE MANUAL FOR:#NOVA-5349-096#NOVA-5349-384A#NOVA-5349-384B#NOVA-5349-384C#NOVA-5349-384D

APPLIED GENOMICS

This product is for research use only.Not for use in diagnostic procedures.

This manual is proprietary to PerkinElmer, Inc., and intended only for customer use in connection with the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose without the prior written consent of PerkinElmer. Follow the protocol included with the kit.

Bioo Scientific, NEXTFLEX, NextPrep, NextPrep-Mag, The NGS Experts, qRNA, Combo-Seq, Sciclone, Zephyr, LabChip, JANUS, and Amplicon Studio are trademarks or registered trademarks of PerkinElmer. All other brands and names contained herein are the property of their respective owners.

PerkinElmer Applied Genomics

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NEXTFLEX® Variant-Seq™ SARS-CoV-2 Kit NOVA-5349-096, NOVA-5349-384A, NOVA-5349-384B, NOVA-5349-384C, and NOVA-5349-384D

GENERAL INFORMATION 4///// Product Overview 4

///// Kit Contents, Storage & Shelf Life 4

///// Required Materials Not Provided 5

///// Warnings & Precautions 5

///// Revision History 5

///// Starting Materials 7

//// Reagent preparation 7

SAMPLE PREP WORKFLOW 6

SAMPLE PREP PROTOCOL 8Step A: Reverse Transcription (cDNA Preparation) 8

Step B: COVID-19 Genome Amplification 8

Step C: Post PCR Size-Selection Cleanup 10

Step D: Fragmentation, End-repair, & Adanylation 11

Step E: Adapter Ligation 12

Step F: Post-Adapter Ligation PCR 14

LIBRARY VALIDATION 16

APPENDIX A 17

Variant-Seq™ SARS-CoV-2 kit | #NOVA-5349-096, #NOVA-5349-384A, #NOVA-5349-384B, #NOVA-5349-384C, #NOVA-5349-384D4

///// Product Overview

Targeting SARS-CoV-2 requires both reverse transcription and whole genome enrichment of the viral RNA. The NEXTFLEX® Variant-Seq™ SARS-CoV-2 kit produces in ~7 hours barcoded, fragmented amplicon libraries that are compatible for fast sequencing using Illumina® platforms. Libraries are constructed using RNA extracted from specimens collected for COVID-19 testing and amplicon material generated from the starting sample RNA input. The kit can be used to prepare single, paired-end, and multiplexed DNA libraries for sequencing using Illumina® platforms. The NEXTFLEX® 1-step Fragmentation, End-Repair, and Adenylation simplifies workflow and shortens hands-on library construction time. In addition, the availability of up to 1,536 different Unique Dual Index adapter barcodes facilitates high-throughput applications.

There are five main steps involved in preparing RNA for sequencing: reverse-transcription, COVID-19 targeted PCR amplification using a design based on the ARTIC V3 primers, fragmentation/end repair/ adenylation, adapter ligation, and post-adapter ligation PCR amplification.

The NEXTFLEX® Variant-Seq™ SARS-CoV-2 kit contains the necessary material to take the user’s extracted nucleic acid sample through preparation and amplification for loading onto flow cells for sequencing.

///// Kit Contents, Storage & Shelf Life

The NEXTFLEX® Variant-Seq™ SARS-CoV-2 kit contains enough material to prepare 96 or 384 samples for Illumina® sequencing. The shelf life of all reagents is at least 6 months when stored properly. The Nuclease-free Water and Resuspension Buffer can be stored at room temperature. The NEXTFLEX® Cleanup Beads XP should be stored at 4°C, and all other components should be stored at -20°C.

Kit Contents 96 rxn 384 rxn

NEXTFLEX® Reverse RT 281 µL 1126 µL

NEXTFLEX® Amplicon PCR Mix (2)1378 µL 11021 µL

NEXTFLEX® Primer Pool 1 442 µL (2)884 µL

NEXTFLEX® Primer Pool 2 442 µL (2)884 µL

NEXTFLEX® Fragmentation Buffer 567 µL (2)1135 µL

NEXTFLEX® Fragmentation Enzyme Mix 288 µL 1152 µL

NEXTFLEX® Ligation Master Mix 1215 µL 4861 µL

NEXTFLEX® PCR Master Mix 1317 µL 5268 µL

NEXTFLEX® Primer Mix 263 µL 1052 µL

Nuclease-free Water 1.5 mL 7 mL

Resuspension Buffer 10 mL 40 mL

NEXTFLEX® Cleanup Beads XP 12 mL 49 mL

NEXTFLEX® Unique Dual Index Barcodes (8 μM, 5 μL per well) 1 plate 4 plates

G E N E R A L I N F O R M A T I O N

perkinelmer-appliedgenomics.com/variant-seq | Support: [email protected] 5

///// Required Materials Not Provided

• 8 µL of nucleic acids extracted from COVID-19 test sample.• Ethanol 80% (room temperature)• 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar• Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)• Magnetic Stand -96 (Thermo Fisher Scientific, Cat # AM10027) or similar• Thermal Cycler• 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes• Nuclease-free barrier pipette tips• Vortex

///// Warnings & Precautions

We strongly recommend that you read the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, contact us at [email protected].

• Do not use the kit past the expiration date.• Try to maintain a laboratory temperature of 20°–25°C (68°–77°F).• Ensure that all pipette tips, microcentrifuge tubes, and other consumables are RNase-free• DTT in buffers may precipitate after freezing. If precipitate is seen, vortex buffer for 1-2 minutes or until the

precipitate is in solution. The performance of the buffer is not affected once the precipitate is in solution.• Ensure pipettes are properly calibrated as library preparations are highly sensitive to pipetting error.• Vortex and micro-centrifuge each component prior to use, to ensure material has not lodged in the cap

or the side of the tube. • Do not remove enzymes from -20ºC until immediately before use; return to -20ºC immediately after use.• Do not freeze NEXTFLEX® Cleanup Beads XP.• Vortex beads until they are a uniform suspension.• Thermal cycling should be performed with a heated lid except where specified.• Maintain a laboratory temperature of 20º–25ºC (68º–77ºF).• Extracted nucleic acids from samples collected for COVID-19 testing will have different amounts of

viral load. When the nucleic acids have been previously assessed with a qPCR assay, the CT value should be between 12-28.

///// Revision History

Version Date Description

V21.02 Februrary 2021 Product Launch

V21.03 March 2021 Product Update

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S A M P L E P R E P W O R K F L O W

= A= N

= T= Adapters with

Cluster Sequence

SEQUENCING(CLUSTER GENERATION)

BEAD CLEANUP (Optional Stopping Point)

DNA

cDNA

FIRST STRAND SYNTHESIS

RNA

ENZYMATIC FRAGMENTATION, END-REPAIR & ADENYLATION

ADAPTER LIGATION & CLEANUP(Optional Stopping Point)

PCR & CLEANUP(Optional Stopping Point)

AMPLIFICATION WITH ARTIC V3 PRIMERS

Figure 1: Sample flow chart with approximate times necessary for each step.


///// Starting Material

The NEXTFLEX® Variant-Seq™ SARS-CoV-2 kit has been optimized and validated using 8 µL of extracted nucleic acids from samples collected for COVID-19 testing. If the nucleic acids have been previously assessed using an RT-PCR assay (such as PerkinElmer® SARS-CoV-2 Real-time RT-PCR Kit), the CT value should be between 12-28.

Please contact [email protected] for additional guidance as kit performance is dependent on quality of extracted nucleic acid sample. This kit will allow you to perform 96 or 384 reactions (see page 4, Warnings and Precautions).

///// Reagent Preparation

1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each NEXTFLEX® component except the NEXTFLEX® Fragmentation Enzyme Mix just prior to use. Nuclease-free Water and Resuspension Buffer should be stored at room temperature. NEXTFLEX® Cleanup Beads XP should be stored at 4°C but equilibrated to room temperature prior to use.

2. DTT in buffers may precipitate after freezing. If precipitate is seen, vortex buffer for 1-2 minutes or until the precipitate is in solution. The performance of the buffer is not affected once the precipitate is in solution.

3. Allow NEXTFLEX® Cleanup Beads XP to come to room temperature and vortex the beads until homogenous.



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

Step B: COVID-19 Genome Amplification

MATERIALS

• NEXTFLEX® Amplicon PCR Mix• NEXTFLEX® Primer Pool 1• NEXTFLEX® Primer Pool 2

User Supplied

• 5 µL of cDNA (from Step A)• Thermal cycler • Nuclease-free PCR Plate • Adhesive PCR Plate Seal

1. For each sample, prepare two separate reactions using the NEXTFLEX® Primer Pool 1 and Pool 2. Assemble all reaction components on ice. NOTE: It is recommended to combine these reagents as a master mix if processing multiple samples.

Step A: Reverse Transcription (cDNA Preparation)

MATERIALS

• NEXTFLEX® Reverse RT

User Supplied

• 8µL of extracted nucleic acid sample from COVID-19 test sample • Thermal cycler • Nuclease-free PCR Plate • Adhesive PCR Plate Seal

1. For each sample, combine the following reagents on ice in a nuclease-free PCR plate:

2. Mix components briefly and spin down if necessary. 3. Apply adhesive PCR plate seal and place in thermal cycler using the following program:

4. Proceed immediately to Step B

L I B R A R Y P R E P P R O T O C O L

2 µL NEXTFLEX® Reverse RT8 µL RNA10 µL TOTAL.

2 min 25oC10 min 55oC1 min 95oCHOLD 4oC


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

Component Reaction 1 Reaction 2NEXTFLEX®

Amplicon PCR Mix 12.5 µL 12.5 µL

NEXTFLEX® Primer Pool 1 4 µL -

NEXTFLEX® Primer Pool 2 - 4 µL

Nuclease free water 6 µL 6 µL cDNA 2.5 µL 2.5 µL

TOTAL 25 µL 25 µL

Step Temperature Time CyclesHeat Activation 98oC 30 sec 1

Denaturation 95oC 15 sec28

Anneal/Extend 63oC 5 minHold 4oC Hold 1

2. Mix thoroughly by pipette and collect all liquid to the bottom of the tube by a quick spin if necessary.

3. Apply adhesive PCR plate seal and place in thermal cycler programed as follows: NOTE: thermal cycler should be preheated at 98°C.

4. Transfer 20 µL of each reaction (reactions 1 and 2) into the new wells for a total of 40 µL.

5. Proceed immediately to Step C.



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

Step C: Post-PCR Size-selection Cleanup

MATERIALS

• NEXTFLEX® Cleanup Beads XP• Resuspension Buffer

User Supplied

• 40 µL pooled reactions from Step B• 80% Ethanol, freshly prepared (room temperature)• Magnetic Stand

1. Add 20 µL of NEXTFLEX® Cleanup Beads XP to each sample. Mix thoroughly until homogenized.

2. Incubate at room temperature for 5 minutes.

3. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes, or until the supernatant appears completely clear.

4. Do not discard the supernatant in this step. Transfer the clear supernatant to a new well. Be careful not to disrupt the magnetic bead pellet or transfer any magnetic beads with the supernatant.

5. Add 20 µL of NEXTFLEX® Cleanup Beads XP to supernatant. Mix thoroughly until homogenized.


7. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes, or until the supernatant appears completely clear.

8. Remove and discard the supernatant. Do not disturb beads. Some liquid may remain in wells.

9. With plate on stand, gently add 200 µL of freshly prepared 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully remove ethanol by pipette.

10. Repeat previous step, for a total of 2 ethanol washes. Ensure all ethanol has been removed.

11. Remove plate from magnetic stand and let dry at room temperature for 3 minutes.

12. Resuspend dried beads with 35 µL of Resuspension Buffer. Mix thoroughly until homogenized.

13. Incubate resuspended beads at room temperature for 2 minutes.

14. Place the 96 well PCR plate on the magnetic stand at room temperature for 2 minutes or until the supernatant appears completely clear.

15. Do not discard the supernatant in this step. Transfer 17.5 uL to fragmentation step below.

16. The procedure may be safely stopped at this step with samples stored at -20oC if needed. To restart the protocol, thaw frozen samples on ice before proceeding.

17. Quantitate your PCR Amplicon pool with Nanodrop and/or Qubit.


µL


2. Apply adhesive PCR plate seal and incubate on a thermal cycler using the following program:

NOTE: The initial 4 °C step is to pre-chill the instrument temperature. Place samples into thermal cycler after the temperature reaches 4 °C and follow the program. For consistent fragmentation, it is recommended to add the enzyme while the samples are in the thermal cycler at the 4C step. NOTE: The final library size will be approximately 120 bp larger than the fragment size.

3. The procedure may be safely stopped at this step with samples stored at -20 °C if needed. To restart the protocol, thaw frozen samples on ice before proceeding to Step E.

Ensure thorough mixing by pipetting up and down. Proceed with adding the enzyme.

NOTE: Do NOT vortex the final NEXTFLEX® Fragmentation reaction. Mix by pipette only. It is important to mix the reaction on ice.

Step D: Fragmentation, End-repair & Adenylation

MATERIALS

• NEXTFLEX® Fragmentation Buffer• NEXTFLEX® Fragmentation Enzyme Mix

User Supplied

• PCR Amplicon Pool (from Step C) • Thermal Cycler• 96 well PCR Plate • Adhesive PCR Plate Seal • Microcentrifuge• Ice

1. For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR plate:

STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

17.5 µL PCR Amplicon Pool5 µL NEXTFLEX® Fragmentation Buffer22.5 µL TOTAL.

1 min 4°C25 min 35°C30 min 65°Cend 4°C

22.5 µL DNA + NEXTFLEX® Fragmentation Buffer mixture2.5 µL NEXTFLEX® Fragmentation Enzyme Mix (DO NOT VORTEX)25 µL TOTAL.



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

Step E: Adapter Ligation

MATERIALS

• NEXTFLEX® Ligation Master Mix• NEXTFLEX® Cleanup Beads XP• NEXTFLEX® Unique Dual Index Barcodes Plate• Nuclease-free Water• Resuspension Buffer

User Supplied

• 25 µL of Fragmented, End Repaired, and Adenylated DNA (from STEP D) • Thermal Cycler• Adhesive PCR Plate Seal• 80% Ethanol, freshly prepared (room temperature)• Magnetic Stand

1. Invert the Ligation Master Mix 5 times to homogenize (DO NOT VORTEX) and place on ice.

2. Each sample will require 2.5 µL of barcoded adapter to be added. Suggestion: To mix, pipette up and down 15 times; visually inspect tubes to ensure proper homogenization. Combine the following in the PCR plate and mix thoroughly by pipette:

Adapter should not be premixed to prevent excess adapter dimer formation.

3. Apply adhesive PCR plate seal and incubate in a thermal cycler with heated lid turned off or open for 15 minutes at 20 °C, followed by a 4 °C hold.

4. Add 30 µL of NEXTFLEX® Cleanup Beads XP to each sample. Mix thoroughly until homogenized.

5. Incubate sample at room temperature for 5 minutes.


7. Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in well.

8. With the plate on the stand, add 200 µL of 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully remove ethanol by pipette.

9. Repeat previous step for a total of 2 ethanol washes. Ensure all ethanol has been removed.


25 µL Fragmented, End Repaired & Adenylated DNA (from Step D)10 µL NEXTFLEX® Ligation Master Mix2.5 µL NEXTFLEX® Barcoded Adapter (8uM)37.5 µL TOTAL


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

10. Remove the 96 well plate from the magnetic stand and let dry at room temperature for 3 minutes.

11. Resuspend dried beads with 12 µL of Resuspension Buffer. Mix thoroughly until homogenized.

12. Incubate sample at room temperature for 2 minutes.


14. Do not discard the sample in this step. Transfer 10 µL of clear sample to a new well. Remove the 96 well PCR plate from the magnetic stand.

15. The procedure may be safely stopped at this step with samples stored at -20 °C if needed. To restart the protocol, thaw frozen samples on ice before proceeding to Step F.



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

Step F: Post-Adapter Ligation PCR

MATERIALS

• NEXTFLEX® PCR Master Mix• NEXTFLEX® Primer Mix• NEXTFLEX® Cleanup Beads XP• Resuspension Buffer

User Supplied

• 10 µL of Adapter Ligated DNA (from STEP E) • Thermal Cycler• Adhesive PCR Plate Seal • 96 Well PCR Plate• 80% Ethanol, freshly prepared (room temperature) • Magnetic Stand

*Thaw NEXTFLEX® PCR Master Mix on ice. Once thawed invert several times or swirl to vigorously mix (DO NOT VORTEX).

1. For each sample, combine the following reagents on ice in the PCR plate. Mix thoroughly.

* These components can be premixed and added in a single step.

2. Apply adhesive PCR plate seal and place in thermal cycler for the following PCR cycles:

3. Add 20 µL of NEXTFLEX® Cleanup Beads XP to each well containing supernatant. Mix thoroughly until homogenized.


5. Place the 96 well PCR plate on the magnetic stand at room temperature for 5 minutes until the supernatant appears completely clear.

6. Remove and discard clear supernatant taking care not to disturb beads. Some liquid may remain in wells.

7. With plate on stand, add 200 μL of 80% ethanol to each magnetic bead pellet and incubate plate at room temperature for 30 seconds. Carefully remove ethanol by pipette.

8. Repeat previous step for a total of 2 ethanol washes. Ensure all ethanol has been removed.

9. Remove plate from magnetic stand and let dry at room temperature for 3 minutes.

10. Resuspend dried beads with 23 μL of Resuspension Buffer.


10 µL Adapter Ligated DNA (from Step E)12.5 µL NEXTFLEX® PCR Master Mix*2.5 µL NEXTFLEX® Primer Mix*25 µL TOTAL

Time Temperature Cycles45 sec 98°C 115 sec 98°C30 sec 60°C 530 sec 72°C1 min 72°C 1

HOLD 12°C 1


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

11. Mix thoroughly until homogenized.

12. Incubate resuspended beads at room temperature for 2 minutes.


14. Do not discard the supernatant in this step. Transfer 20 μL of clear sample to a new well.

15. Remove the 96 well PCR plate from the magnetic stand.

16. Examine your library by electrophoresis to ensure proper library sizing and to verify exclusion of contaminating small and large fragments [recommended: LabChip® GX Touch™ instrument (PerkinElmer)].

17. qPCR is recommended to quantify DNA library templates for optimal cluster density.This can be performed using any qPCR quantification kit for Illumina® platforms and the NEXTFLEX® Primer Mix XP as needed.

18. The library is now ready for cluster generation per the standard Illumina® protocol.Proceed to cluster generation or seal with adhesive PCR plate seal and store at - 20 °C.

NOTE: For sequencing we recommend using a minimum 1x36, aiming for a minimum of 1M clusters/sample (1 million reads/sample if working in single-read mode).



L I B R A R Y V A L I D A T I O N

A)

B)

Figure 2: Example of Optional Assessment of Amplicon Size Prior to Library Construction

Figure 3: Library Validation

Libraries were analyzed using the LabChip® GX Touch™ instrument (PerkinElmer).

A) Fully constructed library of CoV+ sample, 200 ng pooled amplicon inputB) Fully constructed library of CoV+ sample, 1200ng pooled amplicon input


A P P E N D I X A

A

B

C

D

E

F

G

H

21 3 4 5 6 7 8 9 10 11 12

91 17 25 33 41 49 57 65 73 81 89

113 19 27 35 43 51 59 67 75 83 91

102 18 26 34 42 52 56 66 74 83 90

124 20 28 36 44 52 60 68 76 84 92

135 21 29 37 45 53 61 69 77 85 93

146 22 30 38 46 54 62 70 78 86 94

157 23 31 39 47 55 63 71 79 87 95

168 24 32 40 48 56 64 72 80 88 96

///// Oligonucleotide Sequences

XXXXXXXX1 denotes the P5 index region of adapter. The index sequences contained in each adapter are listed below.

XXXXXXXX2 denotes the P7 index region of the adapter. The index sequences contained in each adapter are listed below.

The complete index sequences can be found under the Technical Resources tab at perkinelmerappliedgenomics.com/1536-udis. When entering index sequences for the Illumina® MiniSeq®, NextSeq®, HiSeq® 3000 or HiSeq® 4000 platforms, enter the P5 Index Reverse Complement. For all other Illumina® platforms, enter the P5 Index in the first column. For additional information, please email [email protected].

Plate Format

96 Unique Dual Index Barcoded Adapters / 1 reaction / wellAssay Plate: Axygen P-96-450V-C; 500 µL 96 well “V” Bottom, ClearHeat Seal: 4titude® Pierce Seal 4ti

NEXTFLEX® Sequence (5'-3')

PCR Primer 1 AATGATACGGCGACCACCGAGATCTACAC

PCR Primer 2 CAAGCAGAAGACGGCATACGAGAT

NEXTFLEX® UDI Barcode

AATGATACGGCGACCACCGAGATCTACACXXXXXXXX1 CACTCTTTCCCTACACGACGCTCTTCCGATCT GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXX2ATCTCGTATGCCGTCTTCTGCTTG


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NEXTFLEX...13. Place the 96 well PCR plate on the magnetic stand at room temperature for 2 minutes or until the supernatant appears completely clear. 14. Do not discard the sample