Hybridize Probes

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This step binds target regions of DNA within the pre-enrichment library with the exome capture probes, and optional mitochondrial genome or custom capture probes.

This protocol allows up to 10 μl probe panels to be added to each hybridization reaction.

Consumables

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EHB2 (Enrich Hyb Buffer 2)

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NHB2 (Hyb Buffer 2 + IDT NXT Blockers) (blue cap)

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Twist BioScience for Illumina Exome 2.5 Panel (green cap)

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[Optional] RSB (Resuspension Buffer)

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[Optional] Twist Bioscience for Illumina Mitochondrial Panel (sold separately)

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[Optional] Illumina Custom Enrichment Panel v2 (sold separately)

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Nuclease-free water

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One of the following containers:

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[Plate] 96-well PCR plate

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[Tube] 8-tube strip

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One of the following seals:

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[Plate] Microseal 'B' adhesive seal

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[Tube] 8-tube strip caps

Preparation

Prepare the following consumables:

Item	Storage	Instructions
EHB2	2°C to 8°C	Bring to room temperature. Vortex to mix. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear.
NHB2 (blue cap)	‑25°C to ‑15°C	Thaw at room temperature. When at room temperature, preheat to 50°C on a microheating system for 5 minutes. Vortex at maximum speed three times for 10 seconds each to resuspend. Centrifuge briefly. Pipette up and down from the bottom of the tube. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear. Use while warm to avoid precipitates from reforming.
Twist BioScience for Illumina Exome 2.5 Panel	‑25°C to ‑15°C	Bring to room temperature. Vortex to mix.
[Optional] Twist Bioscience for Illumina Mitochondrial Panel	-25°C to -15°C	Bring to room temperature. Vortex to mix.
[Optional] Illumina Custom Enrichment Panel v2	-25°C to -15°C	Bring to room temperature. Vortex to mix.
EEW (amber tube)	‑25°C to ‑15°C	Bring to room temperature if you are proceeding to Capture Hybridized Probes immediately after the 1.5-hour hold in the IEE-HYB program. If you are extending the hold time, bring to room temperature at least 2 hours before the IEE-HYB program ends.
SMB3	2°C to 8°C	Bring to room temperature if you are proceeding to Capture Hybridized Probes immediately after the 1.5-hour hold in the IEE-HYB program. If you are extending the hold time, bring to room temperature at least 2 hours before the IEE-HYB program ends.

Save the following IEE (Illumina Exome Enrichment)-HYB program.

The IEE-HYB protocol has been optimized and validated using Bio-Rad C1000 Touch Thermal Cycler with 96–Deep Well Reaction Module or Bio-Rad DNA Engine Tetrad 2. For more information, refer to the equipment table in User-Supplied Consumables & Equipment. Alternatively, use a thermal cycler that meets the following criteria:

• Heated Lid

• Minimum temperature control range of 10°C to 98°C

• Minimum temperature accuracy of ±0.25°C

• Maximum reaction volume of 100 µl

• Compatible with full-skirted 96-well PCR plates (automation-friendly)

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Choose the preheat lid option and set to 100°C

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Set the reaction volume to 100 µl.

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98°C for 5 minutes

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18 cycles of 1 minute each, starting at 97°C for the first cycle, then decreasing 2°C per cycle (the 18th cycle is at 63°C)

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Hold for 1.5 hours at 62°C.

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[Optional] For slight performance improvements or convenience, the hybridization hold at 62°C can be increased from 1.5 to 16 hours.

Total running time is ~2 hours.

Procedure

[Optional] Dilute Twist Bioscience for Illumina Mitochondrial Panel.

Mitochondrial DNA (ChrM) is present in greater abundance relative to nuclear DNA in the cell, therefore the Mitochondrial Panel may be diluted before use. A dilution series of the Mitochondrial Panel is recommended before a spike-in to the Exome 2.5 Panel to achieve the desired coverage ratio. The appropriate dilution should be empirically determined for the application and sample type.

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The Mitochondrial Panel can either be used as a single spike-in with the Exome 2.5 Panel, or as a double spike-in with both the Exome 2.5 Panel and a custom panel. Mitochondrial Panel dilution recommendations are provided for each scenario. After the appropriate dilution is determined, the diluted Mitochondrial Panel can be added to the hybridization reaction as described in step 2.

The following tables provide examples of dilution series and the resultant sequencing coverage. Water or RSB may be used as the diluent.

Serial Dilutions of Mitochondrial Panel for Single Spike-In
Dilution	Description	Mito Panel Starting Concentration	Mito Panel Final Concentration	Dilution Factor	Volume (µl) of Mito Panel	Volume (µl) of Diluent
1	1:10 Dilution	Stock	1:10	10	5	45
2	1:100 Dilution	1:10	1:100	10	5	45
3	1:500 Dilution	1:100	1:500	5	10	40
4	1:1000 Dilution	1:500	1:1000	2	25	25

Mitochondrial Genome: Exome Coverage Ratios for Single Spike-In*
Pool	Mito: Exome Panel Ratio	Exome Mean Target Coverage	chrM Mean Target Coverage	chrM / Exome Coverage
1	1:1	61	4574	75
2	1:10	62	921	15
3	1:100	63	239	4
4	1:500	64	167	3
5	1:1000	63	155	2

* Example of empirical data showing the coverage ratio for various Mitochondrial Panel dilutions. Data in Mitochondrial Genome: Exome Coverage Ratios was generated using human cell line DNA from the Coriell Institute (NA24143, NA24149, and NA24385). Enrichment for each pool was performed in 12-plex. Sequencing was performed on a NovaSeq 6000 using the S4 Reagent Kit v1.5. Analysis was performed in BaseSpace Sequence Hub. All fastqs were downsampled to 50M total reads using FASTQ Toolkit Version 2.2.5 and enrichment analysis was performed using DRAGEN Enrichment Version 4.0.3.

Serial Dilutions of Mitochondrial Panel for Double Spike-In
Dilution	Description	Mito Panel Starting Concentration	Mito Panel Final Concentration	Dilution Factor	Volume (µl) of Mito Panel	Volume (µl) of Diluent
1	1:5 Dilution	Stock	1:5	5	5	20
2	1:50 Dilution	1:5	1:50	10	5	45
3	1:250 Dilution	1:50	1:250	5	10	40
4	1:500 Dilution	1:250	1:500	2	25	25

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

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

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Pre-enrichment library pool (30 µl)

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Twist BioScience for Illumina Exome 2.5 Panel (4 μl)

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[Optional] Diluted Twist Bioscience for Illumina Mitochondrial Panel (X μl)

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[Optional] Illumina Custom Enrichment Panel v2 (X μl)

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Nuclease-free water (X µl)

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NHB2 (blue cap) (50 µl)

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EHB2 (10 µl)

Spike-In Options	Reagent Volume (X μl)
Spike-In Options	Exome 2.5 Panel	Diluted Mitochondrial Panel	Custom Enrichment Panel v2	Nuclease-free water
Exome 2.5 Panel only	4	N/A	N/A	6
Single spike-in with Mitochondrial Panel	4	4	N/A	2
Single spike-in with Custom Enrichment Panel v2	4	N/A	4	2
Double spike-in with Mitochondrial Panel and Custom Enrichment Panel v2	4	2*	4	N/A

* The dilution of the Mitochondrial Panel probes should be adjusted for a final volume of 2 μl. Refer to the table Serial Dilutions of Mitochondrial Panel for Double Spike-In.

Using a pipette set to 90 µl, pipette each well 10 times to mix.

Centrifuge as follows.

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[Plate] Seal the plate with Microseal 'B' and centrifuge at 280 × g for 30 seconds.

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[Tube] Cap the tubes and centrifuge at 280 × g for 30 seconds.

Place the sample plate or tubes on the preprogrammed thermal cycler and run the IEE (Illumina Exome Enrichment)‑HYB program.

Proceed immediately to the next procedure when the IEE (Illumina Exome Enrichment)‑HYB program hold temperature time ends.

Precipitation occurs if the temperature of the hybridization reaction falls below room temperature. Proceed directly from Hybridization to Capture Hybridized Probes.