GenieSeq Universal Plus DNA Library Prep Kit (Illumina) (MORV0071)

Components

The Control DNA is derived from salmon genomic DNA and should be re-quantified before use. Not supplied: DNA clean-up beads such as GeniePure DNA Clean-up Beads, Illumina adapters, absolute ethanol, ddH2O, low-binding tubes, PCR tubes, a magnetic rack and a PCR instrument.

Storage

Store at -30 ~ -15°C.

Workflow: fragmentation, end repair and dA-tailing (50 μl) → adapter ligation (100 μl) → 0.6× bead clean-up (PCR-free libraries end here) → library amplification (50 μl) → 0.9× bead clean-up → QC. Optional size selection can be done after ligation or after amplification.

1. Fragmentation, end repair and dA-tailing
   • Dissolve DNA in ddH₂O where possible. The enzyme mix is sensitive to EDTA: if the DNA solvent contains EDTA, either purify the DNA with 2.2× beads and elute in ddH₂O, or add Neutralization Buffer according to the final EDTA concentration in the 50 μl reaction (final EDTA = EDTA in DNA solution × DNA volume / 50 μl; e.g. 10 μl DNA in TE with 1 mM EDTA gives 0.2 mM).

• Thaw FEA Buffer V2 and FEA Enzyme Mix V2, mix, spin down and keep on ice. Work on ice for all set-up steps.
• In a PCR tube combine Input DNA (x μl), Neutralization Buffer (y μl, only if EDTA is present), 5 μl FEA Buffer V2 and ddH₂O to 40 μl.
• Add 10 μl FEA Enzyme Mix V2 last, mix by pipetting or vortexing, spin down and place in the PCR instrument immediately.
• Run: heated lid 105°C; 37°C for the fragmentation time below; 65°C 30 min; 4°C hold.

Times are based on 100 ng high-quality human gDNA in ddH₂O. Within 100 pg - 1 μg input, the same time gives a similar size distribution. For poor-quality DNA or sizes outside the expected range, adjust by 2–5 min. For FFPE DNA, no time adjustment is needed unless insert size is critical.

2. Adapter ligation
   • Dilute adapters according to input so that a fixed 5 μl is added:

• Thaw Rapid Ligation Buffer V2 and Rapid DNA Ligase V2, mix, spin down and keep on ice. A white precipitate in the buffer is normal and dissolves on thawing.
• Combine 50 μl fragmentation product, 25 μl Rapid Ligation Buffer V2, 5 μl Rapid DNA Ligase V2, 15 μl ddH₂O and 5 μl adapter (100 μl total). Mix and spin down.
• Run: heated lid 105°C; 20°C 15 min; 4°C hold. For very low input, the ligation time can be doubled, though this may increase adapter dimers.
• Clean up with 60 μl beads (0.6×): incubate 5 min at room temperature, separate on a magnet (about 5 min) and discard the supernatant; wash twice with 200 μl fresh 80% ethanol (30 sec each); air-dry until the beads turn from shiny to matte brown.
• Elute in 25 μl eluent (10 mM Tris-HCl, pH 8.0–8.5) or ddH₂O and transfer 20 μl. If size selection follows, elute in 105 μl and transfer 100 μl. Store at -20°C.

3. Library amplification
   • Required for non-full-length (stubby) adapters; recommended for full-length adapters when input is <50 ng. With full-length adapters and ≥50 ng input, this step can be skipped for a PCR-free library.
   • Combine 20 μl purified ligation product, 5 μl PCR Primer Mix 3 for Illumina and 25 μl GenieSeq HiFi Amplification Mix (50 μl total). For stubby adapters, use the index primers supplied with the adapter kit instead.
   • Run: 95°C 3 min; then cycles of 98°C 20 sec, 60°C 15 sec, 72°C 30 sec (cycle number below); 72°C 5 min; 4°C hold.

Based on high-quality mouse gDNA fragmented for 15 min at 37°C. Use more cycles for poor-quality DNA or if size selection is performed. Stubby adapters need at least 2 extra cycles.

• Clean up with 45 μl beads (0.9×) as above, air-dry 5–10 min, elute in 25 μl eluent or ddH₂O (ddH₂O if targeted capture follows) and transfer 20 μl. Store at -20°C.

4. Library quality control
   • Check size distribution on an electrophoresis-based instrument (e.g. Bioanalyzer, TapeStation, LabChip GX or Fragment Analyzer).
   • Quantify with a dsDNA fluorescent dye (e.g. GenieQuant 1× dsDNA HS Assay Kit) or by qPCR. qPCR (e.g. GenieQuant Library Quantification Kit for Illumina 2.0) is recommended for PCR-free and over-amplified libraries.

Two-round size selection (optional)

• Perform size selection only once. If input is ≥100 ng, select after adapter ligation; if <100 ng or copy number is limited, select after amplification. Do not omit the post-ligation clean-up before selecting, as the PEG in the ligation buffer interferes.
• Bring the sample to 100 μl with ddH₂O. Add X μl beads, incubate 5 min, separate and transfer the supernatant to a new tube (discard beads). Add Y μl beads, incubate 5 min, separate and discard the supernatant. Wash twice with 200 μl 80% ethanol, air-dry 3–5 min, elute in 25 μl and transfer 20 μl.

Bead-based selection is ineffective for inserts >700 bp; use gel extraction instead. Conditions may need optimising with other bead brands.

Q1: My DNA is dissolved in TE buffer. Can I still use it?

• Yes, but EDTA inhibits the fragmentation enzyme. If the final EDTA concentration in the 50 μl reaction exceeds 0.1 mM, either purify the DNA with 2.2× beads and elute in ddH₂O, or add Neutralization Buffer according to the EDTA table (e.g. 1 μl for 0.2 mM, 5 μl for 1 mM). Do not add excess Neutralization Buffer, as it can cause over-fragmentation.

Q2: How do I control insert size?

• Adjust the 37°C fragmentation time: 20–30 min for 150 bp, 8–12 min for 250 bp, 5–10 min for 350 bp and ≤5 min for 550 bp. If results fall outside the expected range or DNA quality is poor, adjust by 2–5 min. For tighter control, add a two-round bead size selection.

Q3: How can I reduce adapter dimers?

• Dilute the adapter according to DNA input, avoid unnecessarily long ligation times, and use the recommended 0.6× post-ligation clean-up. If dimers remain, bring the purified product to 50 μl with ddH₂O and repeat clean-up with 50 μl (1×) beads; reducing adapter amount may also help.

Q4: Why does my library show a high-molecular-weight tail or smear?

• This is usually over-amplification, where primers run out and non-complementary strands cross-anneal. These products are single-stranded libraries of correct length and sequence normally, but dsDNA dye assays underestimate their concentration, so quantify by qPCR. Reduce the cycle number next time; size selection is not recommended to remove the tail.

Q5: Can I make PCR-free libraries?

• Yes. When ligating full-length adapters with ≥50 ng input and sufficient library yield, the amplification step can be omitted. Recommended input for PCR-free WGS is ≥50 ng without size selection or ≥200 ng with size selection. Quantify PCR-free libraries by qPCR.

Q6: When should I perform size selection?

• Perform it only once, as repeated selection reduces complexity and yield. With ≥100 ng input, select after adapter ligation (after the bead clean-up); with <100 ng or limited sample, select after library amplification. With low input, skipping size selection preserves complexity.