Protocol Checklist

1. Add 2–30 µl DNA to a 96-well PCR plate.
2. If DNA volume < 30 µl, add nuclease-free water to bring the volume to 30 µl.
3. Vortex eBLT (yellow cap) vigorously to resuspend.
4. For each sample, combine the following volumes to prepare the Tagmentation Master Mix.
• eBLT (11.5 µl)
• TB1 (11.5 µl)

Reagent overage is included in the volume to ensure accurate pipetting.

5. Vortex the Tagmentation Master Mix to resuspend.
6. Divide the Tagmentation Master Mix volume into an 8-tube strip.
7. Transfer 20 μl Tagmentation Master Mix to the plate containing a sample.
8. Discard the 8-tube strip.
9. Pipette 10 times or shake at 1600 rpm for 1 minute.
10. Place on the thermal cycler and run the TAG program.
11. Wait until the TAG program has reached the hold temperature before proceeding.
1. Let the plate stand at room temperature for 2 minutes.
2. Add 10 µl ST2 (red cap).
3. Pipette 10 times or shake at 1600 rpm for 1 minute.
4. Seal the plate and incubate at room temperature for 5 minutes.
5. Remove and discard supernatant.
6. Remove from the magnetic stand and add 100 µl TWB.
7. Pipette 10 times or shake at 1600 rpm for 1 minute.
8. Repeat step 4 two times for a total of three washes.
9. Seal the plate and place on the magnetic stand until the liquid is clear.
1. Combine the following volumes to prepare the PCR Master Mix.
• EPM (23 µl)
• Nuclease-free water (23 µl)

Reagent overage is included in the volume to ensure accurate pipetting.

2. Vortex, and then centrifuge the PCR Master Mix at 280 × g for 10 seconds.
3. With the plate on the magnetic stand, remove and discard supernatant.
4. Remove from the magnetic stand.
5. Add 40 µl PCR Master Mix.
6. Immediately pipette 10 times or shake at 1600 rpm for 1 minute.
7. Centrifuge at 280 × g for 3 seconds.
8. Centrifuge the index adapter plate at 1000 × g for 1 minute.
9. Prepare the index adapter plate.
10. Add 10 µl Index 1 (i7) and Index 2 (i5) index adapters.
11. Pipette 10 times or shake at 1600 rpm for 1 minute.
12. Centrifuge at 280 × g for 30 seconds.
13. Place on the thermal cycler and run the eBLT PCR program.

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Shake the plate at 1800 rpm for 1 minute.
2. Place on the magnetic stand until the liquid is clear.
3. Transfer 45 µl supernatant to a new MIDI plate.
4. Resuspend Illumina Purification Beads.
5. For gDNA, blood, or saliva, perform the following steps.
a. Add 77 µl nuclease-free water.
b. Add 88 µl Illumina Purification Beads.
c. Pipette 10 times or shake at 1800 rpm for 1 minute.
d. Seal the plate and incubate for 5 minutes.
e. Place on the magnetic stand until the liquid is clear.
f. beads
g. Transfer 200 µl supernatant to the new MIDI plate containing 20 µl Illumina Purification Beads.
h. Pipette 10 times or shake at 1800 rpm for 1 minute.
i. Discard the first plate.
6. For extracted FFPE, perform the following steps.
a. Add 81 µl Illumina Purification beads.
b. Pipette 10 times or shake at 1800 rpm for 1 minute.
7. Incubate at room temperature for 5 minutes.
8. Place on the magnetic stand until the liquid is clear.
9. Remove and discard supernatant.
10. Wash two times with 200 µl fresh 80% EtOH.
11. Remove and discard residual EtOH.
12. Air-dry for 5 minutes.
13. Remove from the magnetic stand.
14. Add 17 µl RSB.
15. Seal the plate, and then shake at 1800 rpm for 2 minutes.
16. Incubate at room temperature for 2 minutes.
17. Place the plate on the magnetic stand until the liquid is clear.
18. Transfer 15 µl supernatant to a new plate.

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Assess quality of 1 µl library or pooled libraries using one of the following methods.
• Add 1 µl RSB to the library or pooled libraries, and then analyze the 2 µl volume using the Advanced Analytical Fragment Analyzer with the HS-NGS High Sensitivity 474 kit.
• Analyze 1 µl library or pooled libraries using the Agilent Technology 2100 Bioanalyzer using a DNA 1000 kit.
1. Record the indexes for the libraries you plan to pool in this step.
2. Pool pre-enriched libraries based on the sample volumes in the following table.

Library Pool Plexity

Each Pre-Enriched Library Volume (µl)

Total Volume (µl)

1-plex

14

30 (with 16 RSB)

12-plex

2.5

30

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Prepare each pre-enriched library.
2. Record the indexes for the libraries you plan to pool in this step.
3. Combine each library in a 1.5 ml microcentrifuge tube into the plexities shown in the following table.

Library Pools

Each Library Sample (ng)

Total DNA Library
Mass (ng)

1

500

500

12

500

6000
4. Perform one of the following based on the total volume of the pooled pre-enriched libraries:
• If pre-enriched library volume = 30  µl, proceed to Hybridize Probes.
• If pre-enriched library volume < 30  µl, add RSB to reach 30  µl total volume.
• If pre-enriched library volume > 30  µl, concentrate the pooled sample.

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Add the following volumes to a new PCR plate or 8-tube strip in the order listed.

Creating a master mix of NHB2 and EHB2 negatively impacts enrichment performance.

• Pre-enriched library sample or pool (30 µl)
• NHB2 (blue cap) (50 µl)
• Enrichment probe panel (10 µl)
• EHB2 (10 µl)
2. Pipette 10 times to mix.
3. Centrifuge at 280 × g for 30 seconds.
4. Place on the thermal cycler and run the NF‑HYB program.
5. Proceed immediately to the next procedure when the NF‑HYB program ends.

Capture

1. Centrifuge at 280 × g for 30 seconds.
2. to the corresponding well of a new MIDI plate or 1.5 ml microcentrifuge tube.
3. Add 250 µl SMB3, and then mix thoroughly as follows.
• [Plate] Shake at 1200 rpm for 4 minutes.
• [Tube] Vortex at high speed 3 times for 10 seconds each.
4. Place on the microheating system and incubate for 15 minutes at:
• [FFPE] 58°C
• [CEX panel] 58°C
• [Somatic variant calling] 58°C
• [All others] 62°C
5. Immediately centrifuge the sample plate or tube at 280 × g for 30 seconds.
6. Immediately place on a magnetic stand until the liquid is clear.
7. Remove and discard all supernatant.

Wash

1. Remove from the magnetic stand.
2. Add 200 µl preheated EEW (amber tube).
• [Plate] Shake at 1800 rpm for 4 minutes.
• [Tube] Vortex at high speed 3 times for 10 seconds each. Do not centrifuge.
3. Place on the microheating system and incubate for 5 minutes at the following temperature:
• [FFPE] 58°C
• [CEX panel] 58°C
• [Somatic variant calling] 58°C
• [All others] 62°C
4. [Tube] Centrifuge briefly.
5. Immediately place on a magnetic stand until the liquid is clear.
6. Remove and discard all supernatant.
7. Repeat steps 1–6 two times for a total of 3 washes.

Transfer Wash

1. Remove from the magnetic stand.
2. Add 200 µl preheated EEW (amber tube).
• [Plate] Shake at 1800 rpm for 4 minutes.
• [Tube] Vortex at high speed 3 times for 10 seconds each. Do not centrifuge.
3. Transfer 200 µl to a new MIDI plate or tube.

Transferring the reagent minimizes carryover of residual reagents that can inhibit downstream PCR.

4. Place on the microheating system and incubate for 5 minutes at the applicable temperature:
• [FFPE] 58°C
• [CEX panel] 58°C
• [Somatic variant calling] 58°C
• [All others] 62°C
5. [Tube] Centrifuge briefly.
6. Immediately place on a magnetic stand and wait until the liquid is clear.
7. Remove and discard all supernatant.
8. Centrifuge at 280 × g for 30 seconds.
9. Place on a magnetic stand for 10 seconds.
10. Remove and discard residual liquid.

Elute

1. Combine the following volumes to prepare an Elution Master Mix.
• EE1 (28.5 µl)
• HP3 (1.5 µl)
2. Vortex, and then centrifuge the master mix at 280 × g for 10 seconds.
3. Remove from the magnetic stand.
4. Add 23 µl Elution Master Mix.
• [Plate] Shake at 1800 rpm for 2 minutes.
• [Tube] Vortex at high speed 3 times for 10 seconds each.
5. Incubate at room temperature for 2 minutes.
6. Centrifuge at 280 × g for 30 seconds.
7. Place on a magnetic stand until the liquid is clear.
8. Transfer 21 µl supernatant to a new 96-well PCR or new 8-tube strip to the corresponding well of a new 96-well PCR plate or to a new 8 tube strip.
9. Add 4 μl ET2 to each well or tube containing 21 μl supernatant.
10. Slowly pipette 10 times.
11. Centrifuge at 280 × g for 30 seconds.
1. Add 5 μl PPC to each well containing a sample.
2. Add 20 µl EPM to each well containing a sample.
• [Plate] Shake at 1200 rpm for 1 minute.
• [Tube] Pipette to mix, and then cap the 8‑tube strip.
3. Centrifuge at 280 × g for 30 seconds.
4. Place on the thermal cycler and run the AMP program.

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Centrifuge at 280 × g for 30 seconds.
2. Transfer 50 µl supernatant.
3. Resuspend IPB.
4. Add 45 µl IPB.
• [Plate] Shake at 1800 rpm for 1 minute.
• [Tube]Vortex at high speed 3 times for 10 seconds each.
5. Incubate at room temperature for 5 minutes.
6. Centrifuge at 280 × g for 1 minute.
7. Place on a magnetic stand until liquid is clear.
8. Remove and discard all supernatant.
9. Wash two times with fresh 200 µl 80% EtOH.
10. Remove and discard residual EtOH.
11. Air-dry for 5 minutes.
12. Remove from the magnetic stand and add 32 µl RSB.
13. Mix thoroughly as follows.
• [Plate] Shake at 1800 rpm for 1 minute.
• [Tube] Vortex at high speed 3 times for 10 seconds each.
14. Incubate at room temperature for 5 minutes.
15. Centrifuge at 280 × g for 30 seconds.
16. Place on a magnetic stand until liquid is clear.
17. Transfer 30 µl supernatant to a new 96-well PCR plate or 1.5 ml microcentrifuge tube.

SAFE STOPPING POINT

If you are stopping, store at ‑25°C to ‑15°C for up to 30 days.

1. Run 1 µl of the enriched libraries using the Qubit dsDNA BR Assay Kit to quantify library concentration.

Total probe molarity proportionally impacts the post-enrichment library yield.

2. Run 1 µl of the pooled library or the individual libraries on the Agilent Technology 2100 Bioanalyzer using a High Sensitivity DNA kit.
1. For sequencing, Illumina recommends setting up a paired‑end run with 101 cycles per read (2 × 101) and 10 cycles per Index Read.
2. Calculate the molarity value of the library or pooled libraries.
3. Using the molarity value, calculate the volumes of RSB and library needed to dilute libraries to the starting concentration.

Sequencing System

Starting Concentration (nM)

Final Loading Concentration (pM)

HiSeq 4000 and HiSeq 3000 Systems

2–3

150–200

iSeq 100 System

2

100

MiniSeq System

2

1.7–1.8

MiSeq System (v3 reagents)

4

10–12

NextSeq 550 and NextSeq 500 Systems

2

1.4–1.5

NovaSeq 6000 System (standard workflow)

2

175–185
4. Dilute libraries using RSB:
• Libraries quantified as a multiplexed library pool—Dilute the pool to the starting concentration.
• Libraries quantified individually—Dilute each library to the starting concentration. Add 10 µl of each diluted library to a tube.
5. Dilute to the final loading concentration by following the denature and dilute instructions for your system.