NEXTFLEX Next Generation Sequencing Hybridization Panels …...• 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar • Nuclease-free low-bind

KIT CONTAINS: 16 BARCODES | 16 RXNS

USE MANUAL FOR:#NOVA-6001-16#NOVA-6002-16#NOVA-6003-16#NOVA-6004-16#NOVA-6005-16

Compatible with Illumina® platforms

NEXTFLEX®

Next Generation Sequencing Hybridization Panels (RUO) (1 ng – 1 µg)

v19.12

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

@PerkinElmer_AG

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APPLIED GENOMICS

NEXTFLEX® NGS Hybridization Panels (RUO) NOVA-6001-16, NOVA-6002-16, NOVA-6003-16, NOVA-6004-16, NOVA-6005-16

GENERAL INFORMATION 4///// Product Overview 4

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

///// Required Materials Not Provided 5

///// Warnings & Precautions 5

///// Starting Materials 6

///// Reagent Preparation 6

SAMPLE PREP WORKFLOW 7

NGS HYBRIDIZATION PANELS LIBRARY PREPARATION 8 Step A: Fragmentation, End-Repair & Adenylation 8

Step B: Adapter Ligation 10

Step C: PCR Amplification 12

Pre-capture Library Validation 13

Target Enrichment Hybridization 14Step D: Hybridize DNA Library Pools to Baits 14

Step E: Prepare Streptavidin Beads 16

Step F: Capture the Hybridized DNA Using Streptavidin Beads 17

Step G: Post-Capture PCR Amplification 18

Post-capture Library Validation 19

APPENDIX A 20///// Oligonucleotide Sequences 20

NGS Hybridization Panels (RUO) | #NOVA-600X-164

///// Product Overview

The NEXTFLEX® NGS Panel contains library prep reagents, index barcodes, blockers, baits, streptavidin-beads, hybridization and wash buffers, and post-capture amplification reagents. The kit is designed for ~2.5 hour pre-capture DNA library construction, with enzymatic fragmentation included, from as little as 1 ng DNA, and optimized for subsequent target enrichment prior to sequencing. The kit can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina® platforms. The recommended minimum input for hybrid capture is 100 ng of starting material into the pre-capture library prep. There are five main steps involved in preparing DNA libraries for target enrichment: DNA extraction, DNA fragmentation, DNA end repair / adenylation, adapter ligation, and PCR amplification. The kit contains the necessary material to take the user’s purified genomic DNA through fragmentation, library preparation and amplification for target enrichment, hybridization, and post-capture sample processing for sequencing.

The Duchenne Muscular Dystrophy panel (RUO, NOVA-6001-16), Lysosomal Storage Disorders panel (RUO, NOVA-6002-16), Target panel 1(endocrine and metabolic disorders, hemoglobinopathies, primary immunodeficiencies) (RUO, NOVA-6003-16), and Target Panel 2(amino acid disorders, fatty acid disorders, organic acid disorders) (RUO, NOVA-6004-16) are < 3.0 Mb while the Core Exome Panel (RUO, NOVA-6005-16) is > 3.0 Mb. Pay special attention to the size of the panel to ensure proper protocol steps are being followed.

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

The NEXTFLEX® NGS Panel contains enough material to prepare 16 DNA samples. The shelf life of all reagents is at least 6 months when stored properly. The Nuclease-free Water, NEXTFLEX® Hyb 1 XP, NEXTFLEX® Hyb 2 XP, NEXTFLEX® Hyb 4 XP, NEXTFLEX® Binding Buffer XP, NEXTFLEX® Wash Buffer 1 XP, and NEXTFLEX® Wash Buffer 2 XP can be stored at room temperature. The NEXTFLEX® Cleanup Beads XP and NEXTFLEX® Streptavidin Beads should be stored at 4°C. Baits should be stored at -80°C, and all other components should be stored at -20°C.

Kit Contents Cap Color Amount (16 rxns) Storage

NEXTFLEX® Fragmentation Buffer CLEAR CAP 80 µL -20°C

NEXTFLEX® Fragmentation Enzyme Mix CLEAR CAP 176 µL -20°C

NEXTFLEX® Ligase Buffer Mix XP PURPLE CAP 712 µL -20°C

NEXTFLEX® Ligase Enzyme XP PURPLE CAP 48 µL -20°C

NEXTFLEX® DNA Barcoded Adapter PURPLE CAP 2.5µL each -20°C

NEXTFLEX® PCR Master Mix XP GREEN CAP 800 µL -20°C

NEXTFLEX® Primer Mix XP (50 µM) GREEN CAP 64 µL -20°C

NEXTFLEX® HYB 1 XP ORANGE CAP 116 µL Room temp

NEXTFLEX® HYB 2 XP RED CAP 6 µL Room temp

NEXTFLEX® HYB 3 XP YELLOW CAP 48 µL -20°C

NEXTFLEX® HYB 4 XP PINK CAP 62 µL Room temp

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

NGS Hybridization Panels (RUO) | Support: [email protected] 5

///// Required Materials Not Provided

• 1 ng - 1 µg of DNA in up to 34 µL nuclease-free water.• Ethanol 80% (room temperature)• 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar• 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar• Nuclease-free low-bind 0.2 mL and 1.7 mL tubes with caps• Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)• Magnetic Stand -96 (Ambion, Cat # AM10027)• Thermal Cycler with heated lid• 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes• Nuclease-free barrier pipette tips• Vortex • Minicentrifuge with adpters for 1.5-1.8 mL and 0.2 mL tubes/strips• Water bath or incubator oven capable of reaching 65°C• Heat block capable of reaching 65°C• SpeedVac (optional)

///// 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, you may contact your local distributor, or contact us at [email protected].

• Do not use the kit past the expiration date.• 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.• Do not heat the DNA Barcode Adapter above room temperature.• It is highly recommended that NEXTFLEX® Primer Mix XP be used during PCR amplification.

Inadvertent use of an incorrect primer sequence can potentially result in elimination of the index.

NEXTFLEX® Universal Oligo 1 (500 µM) WHITE CAP 20 µL -20°C

NEXTFLEX® Barcode Blockers (250 µM) BLUE CAP 1.2 µL each -20°C

NEXTFLEX® Block Mix (Concentrate) GREEN CAP 51 µL -20°C

NEXTFLEX® RNase Block XP PURPLE CAP 9 µL -20°C

NEXTFLEX® Baits (<3.0 Mb or >3.0 Mb) ORANGE CAP 34 µL or 85 µL -80°C

NEXTFLEX® Cleanup Beads XP BROWN CAP (2) 1.5 mL 4°C

NEXTFLEX® Streptavidin Beads CLEAR CAP 1 mL 4°C

Nuclease-free Water CLEAR CAP BOT. 5 mL Room temp

NEXTFLEX® Binding Buffer XP CLEAR CAP BOT. 18 mL Room temp

NEXTFLEX® Wash Buffer 1 XP CLEAR CAP BOT. 5 mL Room temp

NEXTFLEX® Wash Buffer 2 XP CLEAR CAP BOT. 15 mL Room temp

mailto:NGS%40perkinelmer.com?subject=NEXTFLEX%20NGS%20Hybridization%20Panels%20Kit%20Support



• The NEXTFLEX® Primer Mix that is included in the NEXTFLEX® NGS Barcodes are NOT compatible with this kit and should NOT be used in place of the Primer Mix XP.

• Maintain a laboratory temperature of 20º–25ºC (68º–77ºF).• DNA sample quality may vary between preparations. It is the user’s responsibility to utilize high quality

DNA. DNA that is heavily nicked or damaged may cause library preparation failure. Absorbance measurements at 260 nm are commonly used to quantify DNA and 260 nm / 280 nm ratios of 1.8 - 2.0 usually indicate relatively pure DNA. Other quantification methods using fluorescent dyes may also be used. The user should be aware that contaminating RNA, nucleotides and single-stranded DNA may affect the amount of usable DNA in a sample preparation.

• Note: The presence of EDTA in the DNA sample can result in larger sized fragments in the final library. We strongly recommend the removal of EDTA from the sample prior to fragmentation. However, if removal is not possible, this effect can be partially mitigated by increasing the fragmentation time. For additional guidance, refer to the manual for the NEXTFLEX® Rapid XP DNA-seq kit (NOVA-5149-01, NOVA-5149-02, NOVA-5149-03).

///// Starting Materials

The NEXTFLEX® NGS Hybridization Panel is intended to be used with high quality genomic DNA inputs ranging from 1 ng - 1 µg. We recommend starting with as much input as possible and suggest a minimum input of 100 ng going into the pre-capture library prep for subsequent target enrichment. This kit will allow you to perform 16 reactions.

///// 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 should be stored at room temperature. NEXTFLEX® Cleanup Beads XP and NEXTFLEX® Streptavidin Beads should be stored at 4°C , but equilibrated to room temperature prior to use. Allow NEXTFLEX® Cleanup Beads XP and NEXTFLEX® Streptavidin Beads to come to room temperature and vortex the beads until homogenous.

2. NEXTFLEX® XT Binding Buffer XP, Wash Buffer 1 XP, Wash Buffer 2 XP, and Nuclease-free Water can be stored at room temperature.

3. 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.


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

ENZYMATIC FRAGMENTATION, END-REPAIR & ADENYLATION(50-80 Minutes)

ADAPTER LIGATION & CLEANUP (45 Minutes){Optional Stopping Point)

OPTIONAL SIZE SELECTION (30 Minutes){Optional Stopping Point)

PCR & CLEANUP (40 - 60 Minutes){Optional Stopping Point)

DNA = N= A= T= Adapters with

Cluster Sequence

COMPLEXITY REDUCTION{SEQUENCE CAPTURE)

PREPARE THE DNA SAMPLE LIBRARY

PROCEED TO SEQUENCING ON ILLUMINA INSTRUMENT

HYBRIDIZE AMPLIFIED SAMPLE LIBRARY

AMPLIFY POST-CAPTURE SAMPLE LIBRARY

QUANTITATE & POOL AN EQUAL MASS OF EACH AMPLIFIED, CAPTURED LIBRARY

AMPLIFY PRE-CAPTURE SAMPLE LIBRARY

Sample Library Constructed with DNA

Barcode Adapter 2


Barcode Adapter 4


Barcode Adapter 5



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step A: Fragmentation, End-Repair & Adenylation

MATERIALS

·CLEAR CAP - NEXTFLEX® Fragmentation Buffer·CLEAR CAP - NEXTFLEX® Fragmentation Enzyme Mix·CLEAR CAP BOTTLE - Nuclease-free Water

User Supplied

• DNA in 34 µL (or less) nuclease-free water • Thermal Cycler• 96 well PCR Plate• Adhesive PCR Plate Seal• Microcentrifuge• Ice

NOTE: The presence of EDTA in the DNA sample can result in larger sized fragments in the final library. We strongly recommend the removal of EDTA from the sample prior to fragmentation. However, if removal is not possible, this effect can be partially mitigated by increasing the fragmentation time.

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

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.

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

_ µL Nuclease-free Water

_ µL DNA (1 ng - 1 µg)

5 µL NEXTFLEX® Fragmentation Buffer

39 µL TOTAL

39 µL DNA + NEXTFLEX® Fragmentation Buffer mixture

11 µL NEXTFLEX® Fragmentation Enzyme Mix (DO NOT VORTEX)

50 µL TOTAL

53-75 MINS20 MINS

TOTAL

NGS HYBRIDIZATION PANELS LIBRARY PREPARATION

1 min 4 °C

See fragmentation table 35 °C

30 min 65 °C

End 4 °C


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

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. A full one- minute incubation at 4 °C is not necessary.

The following table lists the recommended incubation times as a guideline for fragmentation. The mode fragment size can be adjusted by changing the duration of incubation at this 35 °C step. These times are recommendations only, and incubation time may need to be optimized for different sample inputs and types to obtain desired mode fragment size.

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 B: Adapter Ligation.

Input DNA

Target Fragment Peak Size

200 - 300 300 - 400 400 - 500 500 - 600

Fragmentation Time (min) at 35 °C

1 ng 24 15 8 410 ng 18 8 5 3

100 ng 15 8 5 3500 ng 15 8 5 3

1000 ng 14 7 5 3



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step B: Adapter Ligation

MATERIALS

·PURPLE CAP - NEXTFLEX® Ligase Buffer Mix XP·PURPLE CAP - NEXTFLEX® Ligase Enzyme XP·PURPLE CAP - NEXTFLEX® DNA Barcoded Adapter (25 µM)·CLEAR CAP BOTTLE - Nuclease-free Water··BROWN CAP - NEXTFLEX® Cleanup Beads XP

User Supplied

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

1. Thaw NEXTFLEX® Ligase Buffer Mix XP to room temperature, and vortex for 5-10 seconds. Do not spin down tube, as this may cause components of the mix to separate and affect performance.

2. The following table lists recommended barcoded adapter concentration dilutions for various input amounts:

Each sample will require 2.5 µL of barcoded adapter to be added. Perform barcoded adapter dilutions if necessary with Nuclease-free Water, depending on input amount and starting barcoded adapter concentration. The following reaction must be mixed thoroughly. The NEXTFLEX® Ligase Buffer Mix XP is very viscous. Thorough mixing of the reaction below is critical to obtaining optimal results. 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:

55 MINS40 MINS

TOTAL

Input DNA Desired Adapter Adapter Dilution Concentration

1 ng 0.3 µM 1 / 8010 ng 0.6 µM 1 / 40

100 ng 6.25 µM 1 / 4250 ng 25 µM None500 ng 25 µM None

1 µg 25 µM None

50 µL Fragmented, End Repaired & Adenylated DNA (from Step A)

44.5 µL NEXTFLEX® Ligase Buffer Mix XP*

2.5 µL NEXTFLEX® Barcoded Adapter

3 µL NEXTFLEX® Ligase Enzyme Mix XP*

100 µL TOTAL

*These components can be premixed and added in a single step. Adapter should not be premixed in order to prevent excess adapter dimer formation.


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

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 65 µL of Nuclease-free Water and 35 µL of NEXTFLEX® Cleanup Beads XP to each sample. Mix thoroughly until homogenized. The NEXTFLEX® Cleanup Beads XP and Nuclease-free Water can be premixed and added in a single step.

5. Incubate sample at room temperature for 5 minutes.

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

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.

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

11. Resuspend dried beads with 28 µL of Nuclease-free water. Mix thoroughly until homogenized.

12. Incubate sample at room temperature for 2 minutes.


14. Do not discard the sample in this step. Transfer 23 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 C: PCR Amplification.



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step C: PCR Amplification

MATERIALS

·GREEN CAP - NEXTFLEX® PCR Master Mix XP·GREEN CAP - NEXTFLEX® Primer Mix XP (50μM) ·CLEAR CAP BOTTLE - Nuclease-free Water··BROWN CAP - NEXTFLEX® Cleanup Beads XP

User Supplied

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

Note: The NEXTFLEX® Primer Mix that is included in the NEXTFLEX® NGS Barcodes are NOT compatible with this kit and should NOT be used in place of the Primer Mix XP.

*The following table lists recommended PCR cycles:

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

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

3. Add 45 µL of NEXTFLEX® Cleanup Beads XP to each sample. Mix thoroughly until homogenized.

0-55 MINS0-30 MINS

TOTAL

Input DNA Number of PCR cycles

1 ng 14 - 1610 ng 10 - 12

100 ng 7 - 8250 ng 5 - 6500 ng 4 - 5

1000 ng 3 - 4

23 µL Adapter Ligated DNA (from Step B)

25 µL NEXTFLEX® PCR Master Mix XP*

2 µL NEXTFLEX® Primer Mix XP*

50 µL TOTAL

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

30 sec 98°C

15 sec 98°CRepeat as suggested in above table30 sec 65°C

30 sec 72°C

2 min 72°C


4. Incubate at room temperature for 5 minutes.


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 33 µL of Nuclease-free Water. Mix thoroughly until homogenized.

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


13. Do not discard the supernatant in this step. Transfer 30 µL of clear sample to a new well. Remove the 96 well PCR plate from the magnetic stand.

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

15. qPCR may be used 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. Alternatively, a fluorometric method may be used instead.

16. The library is now ready for hybridization enrichment or seal with adhesive PCR plate seal and store at - 20 °C.

Pre-capture Library Validation

Figure 1: Library electropherogram trace

250 ng input, 6 cycle PCR product diluted 1/20 prior to loading.

STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step D: Hybridize DNA Library Pools to Baits

MATERIALS

·ORANGE CAP - NEXTFLEX® HYB 1 XP·ORANGE CAP - NEXTFLEX® Baits·RED CAP - NEXTFLEX® HYB 2 XP·YELLOW CAP - NEXTFLEX® HYB 3 XP·PINK CAP - NEXTFLEX® HYB 4 XP·GREEN CAP - NEXTFLEX® Block Mix (Concentrate) ·WHITE CAP - NEXTFLEX® Universal Oligo 1 (500 µM) ·BLUE CAP - NEXTFLEX® Barcode Blockers (250 µM) ·PURPLE CAP - NEXTFLEX® RNase Block XP

User Supplied

• DNA Libraries (from Step C)• Snap-top centrifuge tubes (optional for SpeedVac only)• Thermal cycler• 96 Well PCR Plate with adhesive seals or strip tubes with caps • Magnetic Stand

This step requires an incubation at 65°C with a heated lid at 105°C for 24 hours. Before performing the hybridization, it is recommended to test that the plate and capping method are appropriate for the thermal cycler to be used. A test run of 27 µL of Nuclease-free Water can be performed to ensure that no more than 4 µL is lost to evaporation under the conditions used for hybridization.

1. Each hybridization reaction requires 750 ng of prepared DNA2. Transfer 750 ng of each Rapid XP library to a separate well of a 96-well plate or capped

strip-tube. Place plate or tubes with wells open in a thermal cycler set to 65°C or use a vacuum concentrator (such as a SpeedVac) to dry samples down completely.

3. While libraries are drying, prepare Block Mix (working solution):

4. Pipet 8.8 µL Block Mix (working solution) plus 1.2 µL Barcode Blockers (250 µM) (total 10 µL) into each well containing a dried library pellet and pipet gently up and down to suspend completely. Transfer sealed plate or strip tubes to the thermal cycler and incubate using the following program:

16-24 HRS60 MINS

TOTAL

T A R G E T E N R I C H M E N T H Y B R I D I Z A T I O N

1 RXN 16 RXN Component

3 µL 51 µL NEXTFLEX® Block Mix (concentrate)

0.6 µL 10.2 µL NEXTFLEX® Universal Oligo 1 (500 µM)

5.2 µL 88.4 µL Nuclease-free Water

8.8 µL 149.6 µL TOTAL

5 min 95°C

Hold 65°C (at least 5 mins)


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G


6.63 µL 116 µL NEXTflex™ HYB 1 XP

0.27 µL 4.7 µL NEXTflex™ HYB 2 XP



13 µL 227.5 µL TOTAL

5. While the Block Mix (working solution) incubates, prepare the Hybridization Buffer by mixing the following components at room temperature:

*Prepare Hybridization Buffer for at least 5 reactions to allow accurate pipetting volumes. If a precipitate forms, warm the Hybridization Buffer at 65°C for 5 minutes. Keep the prepared Hybridization Buffer at room temperature until it is used in later steps.Use a heated lid set at 105°C to hold the temperature at 65°C. Make sure that the DNA sample and Block Mix are held at 65°C for at least 5 minutes before adding the remaining hybridization reaction components below.

6. Prepare the appropriate dilution of NEXTFLEX® RNase Block XP, based on the size of your Capture Library Baits. Keep the mixture on ice until it is used in subsequent steps.For Capture Libraries < 3.0 Mb, make a 10% dilution of the RNase Block XP by adding 1 part RNase Block to 9 parts Nuclease-free Water. A total of 5 µL is needed for each target capture reaction.

For Capture Libraries ≥ 3.0 Mb, make a 25% dilution of the RNase Block by adding 1 part RNase Block XP to 3 parts Nuclease-free Water. A total of 2 µL is needed for each target capture reaction.

7. Prepare the Capture Library Hybridization Mix appropriate for your Capture Library Baits Size. Mix well by vortexing at high speed for 5 seconds then spin down briefly. Keep the mixture at room temperature until use in the next step.

8. Combine Hybridization Buffer, RNAse Block XP, and Capture Libray for final Hybridization Mix:


0.5 µL 8.5 µL NEXTFLEX® RNase Block XP


5 µL 85 µL TOTAL


0.5 µL 8.5 µL NEXTFLEX® RNase Block XP


2 µL 34 µL TOTAL



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

For Capture Libraries < 3.0 Mb

For Capture Libraries ≥ 3.0 Mb

9. Maintain the gDNA library & Block Mix plate or strip tube at 65°C while you add 20 µL of the Capture Library Hybridization Mix to each well containing DNA library and Block Mix. Mix well by pipetting. The hybridization reaction wells now contain approximately 28 µL - 30 µL, depending on the degree of evaporation during the Block Mix incubation.

10. Apply adhesive seal or strip caps. Make sure that all wells are completely sealed. It is crucial to limit evaporation at this stage.

11. Incubate the hybrdization reaction for 16 - 24 hours at 65°C with a heated lid at 105°C. Proceed to Step E: Prepare Streptavidin Beads.

Step E: Prepare Streptavidin Beads

MATERIALS

·CLEAR CAP - NEXTFLEX® Streptavidin Beads·CLEAR CAP BOTTLE - NEXTFLEX® Binding Buffer XP

User Supplied

• 96 Well PCR Plate• Magnetic Stand

1. Vigorously resuspend the NEXTFLEX® Streptavidin Beads by vortexing.2. Per each target capture reaction, add 50 µL of the resuspended beads to a fresh PCR plate.3. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes.4. Remove and discard clear supernatant.5. Resuspend beads with 200 µL NEXTFLEX® Binding Buffer XP, mixing throughly by

pipetting.6. Incubate resuspended beads at room temperature for 2 minutes.7. Place the 96 well PCR Plate on the magnetic stand at room temperature until superna-

tant has completely cleared.8. Repeat steps 5 - 8 for a total of three washes.9. Remove and discard clear supernatant.10. Resuspend beads with 200 µL NEXTFLEX® Binding Buffer XP, mixing thoroughly by

pipetting.11. Proceed to Step F: Capture the Hybridized DNA Using Streptavidin Beads.

40 MINS40 MINS

TOTAL


13 µL 221 µL Hybridization Buffer mixture from Step 5

5 µL 85 µL 10% RNase Block solution from Step 6

2 µL 34 µL Capture Library Baits

20 µL 340 µL TOTAL


13 µL 221 µL Hybridization Buffer mixture from Step 5

5 µL 85 µL 10% RNase Block solution from Step 6

2 µL 34 µL Capture Library Baits

20 µL 340 µL TOTAL


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step F: Capture the Hybridized DNA Using Streptavidin Beads

MATERIALS

·CLEAR CAP BOTTLE - NEXTFLEX® Wash Buffer 1 XP·CLEAR CAP BOTTLE - NEXTFLEX® Wash Buffer 2 XP

User Supplied

• Hybridized DNA Library Pools (from Step D)• Washed Streptavidin Beads (from Step E)• Nutator mixer or equivalent• Thermal cycler• 96 Well PCR Plate and strip tube caps• 80% Ethanol, freshly prepared (room temperature)• Magnetic Stand• Ice

1. Prewarm the NEXTFLEX® Wash Buffer 2 XP by pipetting 200 µL per capture in triplicate into a 96-well PCR plate and place the sealed plate in a thermal cycler set to 65°C. Leave there for step 12.

2. Make sure that the volume of hybridization reaction that remains is over 24 µL after the incubation.

3. Maintain the hybridization plate at 65°C while transferring the entire volume of each hybridization mixture to the plate wells containing 200 µL of washed streptavidin beads. Mix well by pipetting.

4. Seal the wells and incubate the capture plate on a Nutator mixer or equivalent for 30 minutes at room temperature. Make sure that samples are properly mixing in the wells.

5. Briefly spin the plate in a centrifuge or mini-plate spinner.6. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes, or

until the supernatant appears completely clear.7. Remove and discard the supernatant.8. Resuspend the beads in 180 µL of NEXTFLEX® Wash Buffer 1 XP. Mix by gentle pipetting

the volume up and down 10 to 20 times. Take care not to create too many bubbles.9. Incubate for 15 minutes at room temperature. Briefly spin in a centrifuge or mini-plate

spinner.10. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes, or

until the supernatant appears completely clear.11. Remove and discard the supernatant.12. It is important to maintain bead suspensions at 65°C during the following washing

procedure to ensure specificity of the capture. Wash the beads with the pre-warmed 65°C NEXTFLEX® Wash Buffer 2 XP as follows:A. Add 200 µL of pre-warmed 65°C NEXTFLEX® Wash Buffer 2 XP and pipet up and

down to suspend bellet pellet.B. Incubate at 65°C for 10 minutes.C. Place the plate on the magnetic stand for for a short time ONLY UNTIL

SUPERNATANT BECOMES CLEAR, discard supernatant, and return plate to thermal cycler (it is important to maintain 65°C).

D. Repeat steps 12:A through C for a total of 3 washes (during final 10-minute incubation, jump to step G:1 - make PCR master mix and keep on ice until ready for use).

13. Keep the samples on ice until they are used in Step G: PCR Amplification.

90 MINS40 MINS

TOTAL



STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

Step G: Post-Capture PCR Amplification

MATERIALS

·GREEN CAP - NEXTFLEX® PCR Master Mix XP·GREEN CAP - NEXTFLEX® Primer Mix XP (50µM) ··BROWN CAP - NEXTFLEX® Cleanup Beads XP·CLEAR CAP BOTTLE - Nuclease-free Water

User Supplied

• Hybridized Library, bound to beads (from Step F)• Thermal cycler• 96 Well PCR Plate• 80% Ethanol, freshly prepared (room temperature)• Magnetic Stand • Ice

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

2. Resuspend Hybridized Libray, bound to beads (from STEP F) with 50 µL PCR mix and perform PCR on-bead.

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

4. Add 45 µL of the NEXTFLEX® Cleanup Beads XP to each sample. Mix thoroughly until homogenized.

5. Incubate at room temperature for 5 minutes.

6. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes.

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

8. 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.

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

50 MINS30 MINS

TOTAL

25 µL NEXTFLEX® PCR Master Mix XP

2 µL NEXTFLEX® Primer Mix XP

23 µL Nuclease-free Water

50 µL TOTAL

30 sec 98°C

15 sec 98°C

Repeat for a total of 12 cycles30 sec 65°C

30 sec 72°C

2 min 72°C


STEP ASTEP B

STEP CSTEP D

STEP ESTEP F

STEP G

10. Remove plate from magnetic stand and let dry at room temperature for 5 minutes or until bead pellet is visibly dry.

11. Resuspend dried beads with 33 µL Nuclease-free water. Mix thoroughly until homogenized.

12. Incubate resuspended beads at room temperature for 5 minutes.

13. Place the 96 well PCR Plate on the magnetic stand at room temperature for 5 minutes.

14. Transfer 30 µL of clear sample to a new well.

15. Examine your library by LabChip® GXII Touch™ HT instrument (PerkinElmer®) or equivalent. Ensure that electropherogram shows no adapter dimers or primer dimers. Assess concentration of samples by Qubit, and proceed to Illumina sequencing preparation.

Post-capture Library Validation

Figure 2: Post-capture library electropherogram trace

750 ng input, 12 cycle PCR product diluted 1/10 prior to loading



A P P E N D I X A

///// Oligonucleotide Sequences

XXXXXXXX1 denotes the index region of the adapter. The index sequence of each adapter is listed below.

XXXXXXXX2 denotes the index region of the barcode blocker. The sequence of each barcode blocker is listed below.

NEXTFLEX® Sequence

Primer 1 5'AATGATACGGCGACCACCGAGATCTACAC

Primer 2 5'CAAGCAGAAGACGGCATACGAGAT

Barcoded Adapter

5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT5'GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXX1ATCTCGTATGCCGTCTTCTGCTTG

NEXTFLEX® Sequence

HEUniversal Oligo 1

5'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

Barcode Blocker

5'CAAGCAGAAGACGGCATACGAGATXXXXXXXX2GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

Index 1 AACGTGATIndex 3 ATGCCTAAIndex 9 CGCTGATC

Index 16 AAGACGGAIndex 22 ACGTATCAIndex 28 ATCCTGTAIndex 51 GCGAGTAAIndex 57 GTCGTAGA

Index 1 ATCACGTTIndex 3 TTAGGCATIndex 9 GATCAGCG

Index 16 TCCGTCTTIndex 22 TGATACGTIndex 28 TACAGGATIndex 51 TTACTCGCIndex 57 TCTACGAC

Index 63 TCTTCACAIndex 72 AATCCGTCIndex 73 AATGTTGCIndex 74 ACACGACCIndex 75 ACAGATTCIndex 76 AGATGTACIndex 77 AGCACCTCIndex 92 GAACAGGC

Index 63 TGTGAAGAIndex 72 GACGGATTIndex 73 GCAACATTIndex 74 GGTCGTGTIndex 75 GAATCTGTIndex 76 GTACATCTIndex 77 GAGGTGCTIndex 92 GCCTGTTC






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Documents

NEXTFLEX Next Generation Sequencing Hybridization Panels …...• 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar • Nuclease-free low-bind