GenieSeq Capture Hybridization & Wash Kit (MORV0076)

Components

Also required: probe panel, universal blockers and post-PCR primer mix matched to the library adapter type, DNA clean-up beads, absolute ethanol, ddH2O, 0.1× TE, elution buffer (10 mM Tris-HCl, pH 8.0–8.5), low-binding 200 μl PCR tubes and 1.5 ml tubes, magnetic stand, vacuum concentrator, PCR instrument, metal bath, vortex mixer, handheld centrifuge, fluorometer and fragment analyser.

Storage

Store at -30 ~ -15°C.

Workflow: library pooling and concentration (45 min) → library and probe hybridisation (4–16 h) → wash buffer and bead preparation, streptavidin bead capture and post-capture wash (1.5 h) → post-capture PCR and purification (1.5 h).

1. Pool and concentrate libraries
   • In a 1.5 ml nuclease-free tube, combine the pre-library (500 ng of each library; ≤5 μg in total when multiplexing), 5 μl Cot-1 DNA and 2 μl Universal Blockers.
   • When multiplexing, use 500–1,000 ng of each library and no less than 125 ng.
   • Choose blockers matching the adapter type: GenieSeq Capture Universal Blockers & Post-PCR Primer Mix for Illumina TruSeq libraries, or the corresponding blockers for MGI single- or dual-indexed libraries.
   • Dry down in a vacuum concentrator (≤50°C). If not hybridising immediately, store at -30 ~ -15°C for no more than 24 h.
2. Hybridise library and probes
   • Thaw the reagents and mix well. Add to the dried library:

• Leave at room temperature for 10 min, mix by pipetting and transfer to a new 200 μl low-binding PCR tube that is airtight and heat-resistant.
• Run: heated lid 100°C on; 95°C for 30 sec; 65°C for 4–16 h.

3. Prepare reagents
   • Dilute wash buffers to 1× as below (volumes per reaction; for N reactions multiply by 1.1 × N). Incubate 10× Wash Buffer I at 65°C for at least 10 min before use to dissolve completely.

• Pre-warm the 1× Wash Buffer I and 1× Wash Buffer S used for heated washes at 65°C for about 20 min; avoid over-warming to prevent evaporation.
• Prepare the Beads Resuspension Mix per reaction in a low-binding tube: 8.5 μl 2× Hybridization Buffer, 2.7 μl Hybridization Enhancer and 5.8 μl nuclease-free ddH₂O (17 μl total).

4. Wash the streptavidin beads
   • Use 100 μl of beads per capture. Equilibrate the beads from 4°C to room temperature and vortex for 15 sec. Do not let the beads dry out.
   • Add 100 μl beads to a 200 μl low-binding PCR tube, place on the magnet until clear and discard the supernatant.
   • Off the magnet, add 200 μl 1× Beads Wash Buffer and vortex for 10 sec; spin briefly, separate on the magnet and discard the supernatant. Repeat once (two washes in total).
   • Resuspend the beads in 17 μl Beads Resuspension Mix.
5. Capture
   • Keep the hybridisation tube in the PCR instrument at all times. Pre-warm the resuspended beads in the instrument (heated lid 100°C) for 1 min, then quickly add them to the hybridisation reaction and pipette 15 times to mix.
   • Incubate at 65°C for 45 min, removing the tube every 15 min to vortex gently. Work quickly; tap the liquid down rather than centrifuging.
6. Post-capture wash
   • Heated wash (lid and block at 65°C): add 100 μl pre-warmed 1× Wash Buffer I, pipette 10 times, separate on the magnet for 30 sec and remove the supernatant. Return the tube to the instrument immediately, add 180 μl pre-warmed 1× Wash Buffer S, pipette 10 times and incubate at 65°C for 5 min, pipetting 10 times every 2.5 min. Spin briefly, separate and remove the supernatant. Repeat the Wash Buffer S wash once (two in total).
   • Room-temperature washes: wash sequentially with 180 μl 1× Wash Buffer I, then 1× Wash Buffer II, then 1× Wash Buffer III. For each, vortex and incubate at room temperature for 2 min, mixing every 30 sec, then spin briefly, place on the magnet for 1 min and remove the supernatant.
   • Resuspend the beads in 20 μl nuclease-free ddH₂O. Do not discard the beads.
7. Post-capture PCR
   • Combine 20 μl resuspended beads, 25 μl 2× GenieSeq HiFi Amplification Mix and 5 μl Post-PCR Primer Mix (matched to the library platform and adapter type). Mix and run:

Recommended number of cycles (N):

Panel, blockers and pre-library insert size all affect yield; adjust the cycle number as needed.

8. Purify the PCR product
   • Add 60 μl GeniePure DNA Clean-up Beads to the 50 μl PCR product, vortex and incubate for 5 min at room temperature. Spin briefly, separate on the magnet (about 5 min) and remove the supernatant.
   • Wash twice with 200 μl freshly prepared 80% ethanol for 30 sec each, keeping the tube on the magnet.
   • Air-dry for 1–3 min until no ethanol remains. Elute in 25 μl nuclease-free ddH₂O, separate (about 5 min) and transfer 20 μl of the supernatant to a new 1.5 ml nuclease-free tube.
9. Library quality control
   • Measure concentration with a dsDNA fluorescent dye assay such as the GenieQuant 1× dsDNA HS Assay Kit, or by qPCR absolute quantification such as the GenieQuant Library Quantification Kit for Illumina 2.0.
   • Check size distribution on an electrophoresis-based fragment analysis system.

Q1: Which libraries and blockers are compatible?

• Use blockers matched to the pre-library adapter type: the GenieSeq Capture Universal Blockers & Post-PCR Primer Mix for Illumina TruSeq libraries, or the corresponding blockers for MGI single- or dual-indexed libraries. Mismatched blockers reduce on-target rate and can cause capture to fail.

Q2: How much library DNA should I use?

• Use 500 ng of each library. When multiplexing, use 500–1,000 ng per library (no less than 125 ng) and no more than 5 μg in total.

Q3: How should I concentrate pooled libraries?

• Dry them down in a vacuum concentrator at ≤50°C. Bead-based concentration may introduce GC bias. If not hybridising immediately, store the concentrated library at -30 ~ -15°C for no more than 24 h.

Q4: How many post-capture PCR cycles should I run?

• For an exome panel (≥30 Mb), 10, 8, 7 or 6 cycles for 1, 4, 8 or 12 pooled libraries; for 3 Mb, 14, 13, 13 or 11; for 0.3 Mb, 15, 14, 14 or 12. Adjust for your panel, blockers and insert size.

Q5: What could cause a low on-target rate or poor uniformity?

• Mismatched blockers and inaccurate temperature control during hybridisation and washing are the main causes. Keep the hybridisation tube in the PCR instrument at 65°C during bead addition and heated washes, and work quickly.