Description
Enzymatic DNA library prep kit for MGI
GenieSeq Universal Plus DNA Library Prep Kit (MGI) is an all-enzymatic DNA library prep kit for the MGI/DNBSEQ platform. It integrates fragmentation and end repair in one step with no mechanical shearing, and is optimised for FFPE DNA and broad input ranges with low chimera rates.
Fragmentation, end repair and dA-tailing take place in a single reaction, and the product goes straight into adapter ligation without purification, followed by library amplification and size selection. 100 pg - 1 μg of DNA is converted into an MGI-ready library, and insert size is set simply by adjusting the fragmentation time. This version greatly reduces artificial inverted chimeric reads in FFPE DNA libraries, lowering false-positive SNV and InDel calls for more reliable variant detection. The kit accepts genomic DNA from animals, plants and microorganisms as well as FFPE DNA, and is recommended for whole genome sequencing and whole exome or other targeted capture sequencing. PCR-free libraries are also possible with full-length adapters. For research use only.
Key Features
| Built for MGI | Produces dedicated libraries for MGI sequencing platforms. | |
| Lower false positives | Fewer chimeric reads in FFPE libraries improve SNV and InDel calling accuracy. | |
| One-step fragmentation | Enzymatic fragmentation, end repair and dA-tailing in one tube with no shearing or clean-up. | |
| Wide input range | Converts 100 pg - 1 μg DNA from diverse sources, including FFPE DNA. | |
| High conversion | Good stability with high library conversion rate and yield. |
Specifications
| Platform | MGI sequencing platforms |
| Input DNA | 100 pg - 1 μg (animal, plant, microbial gDNA; FFPE DNA) |
| Fragmentation | Enzymatic, 37°C for 5–30 min (insert size 150–550 bp) |
| Workflow | Fragmentation/end repair/dA-tailing → adapter ligation → amplification (optional) → clean-up |
| Applications | Whole genome sequencing; whole exome and targeted capture sequencing; PCR-free libraries |
| Shelf life | 2 years |
Components
| Components | 24 rxns | 96 rxns |
| FEA Buffer V2 | 120 μl | 480 μl |
| FEA Enzyme Mix V2 | 240 μl | 960 μl |
| Rapid Ligation Buffer V2 | 600 μl | 4 × 600 μl |
| Rapid DNA Ligase V2 | 120 μl | 480 μl |
| GenieSeq HiFi Amplification Mix | 600 μl | 4 × 600 μl |
| PCR Primer Mix for MGI | 120 μl | 480 μl |
| Neutralization Buffer | 120 μl | 480 μl |
| Control DNA (100 ng/μl) | 10 μl | 10 μl |
The Control DNA is derived from the salmon genome and should be re-quantified before use.
Storage
Store at -30 ~ -15°C.
Materials required
- Magnetic beads: GeniePure DNA Clean-up Beads.
- DNA quality control: Agilent 2100 Bioanalyzer or equivalent; GenieQuant 1× dsDNA HS Assay Kit.
- DNA adapters for MGI (full-length or stubby dual-index adapters).
- Absolute ethanol, ddH2O, low-adsorption tubes, PCR tubes, magnetic rack and PCR instrument.
Recommended input DNA
| Application | Sample type | Recommended input |
| Whole genome sequencing | Complex gDNA | 50 ng - 1 μg |
| Targeted capture sequencing | Complex gDNA | 10 ng - 1 μg |
| Whole genome/targeted capture sequencing | FFPE DNA | ≥50 ng |
| Whole genome sequencing | Microbial genome | 1 ng - 1 μg |
| Whole genome sequencing (PCR-free) | Complex/simple genome | ≥50 ng (without size selection); ≥200 ng (with size selection) |
Use high-quality DNA (A260/A280 = 1.8–2.0); increase input for poor-quality DNA.
- Fragmentation, end repair and dA-tailing
- Dissolve DNA in ddH2O where possible; FEA Enzyme Mix V2 is sensitive to EDTA. If the DNA contains EDTA, purify it with 2.2× beads and elute in ddH2O, or add Neutralization Buffer according to the final EDTA concentration in the 50 μl reaction (EDTA in DNA solution × DNA volume / 50 μl): 1 mM, 5 μl; 0.8 mM, 4 μl; 0.6 mM, 3 μl; 0.5 mM, 2.5 μl; 0.4 mM, 2 μl; 0.2 mM, 1 μl; 0.1 mM, 0.5 μl; <0.1 mM, 0 μl. Too much Neutralization Buffer causes over-fragmentation.
- Thaw FEA Buffer V2 and FEA Enzyme Mix V2, mix, spin down and keep on ice. Work on ice.
- Combine input DNA (x μl), Neutralization Buffer (y μl, if needed) and 5 μl FEA Buffer V2, and make up to 40 μl with ddH2O.
- Add 10 μl FEA Enzyme Mix V2 last, mix by pipetting or vortexing, spin down and start the program immediately.
- Program (heated lid 105°C): 37°C for the fragmentation time below, 65°C for 30 min, 4°C hold.
| Expected insert size | Fragmentation time at 37°C |
| 150 bp | 25–30 min |
| 250 bp | 17–22 min |
| 350 bp | 10–15 min |
| 550 bp | 5–10 min |
Times are based on 100 ng high-quality human gDNA in ddH2O; inputs across 100 pg - 1 μg give similar distributions. For poor-quality DNA or off-target sizes, adjust by 2–5 min. For FFPE DNA without strict insert-size requirements, no adjustment is needed.
- Adapter ligation
- Dilute the adapter for your input so that a fixed 5 μl is added: 1 ng, 0.1 μM; 10 ng, 1 μM; 50 ng, 5 μM; 100 ng - 1 μg, 10 μM. Avoid repeated freeze-thaw of adapters.
- Thaw Rapid Ligation Buffer V2 and Rapid DNA Ligase V2, mix, spin down and keep on ice. 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 DNA adapter. Mix and spin down.
- Incubate at 20°C for 15 min (heated lid 105°C), then hold at 4°C. For very low inputs, the ligation time can be doubled, though this may increase adapter dimers.
- Clean up with 60 μl beads (0.6×) per 100 μl ligation: incubate 5 min at room temperature, separate on the magnet (about 5 min), discard the supernatant, wash twice with 200 μl fresh 80% ethanol (30 sec each) and 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 recover 20 μl; or, if two-round size selection follows, elute in 105 μl and recover 100 μl.
- Safe stopping point: store at -20°C. A PCR-free library is complete at this stage.
- Library amplification
- Required for stubby adapters; recommended for full-length adapters when input is <50 ng. With ≥50 ng input, or if amplification is not needed, skip to quality control.
- Combine 20 μl purified ligation product, 5 μl PCR Primer Mix for MGI (or the matching index primers for stubby adapters) and 25 μl GenieSeq HiFi Amplification Mix (50 μl total). Mix and spin down.
| Step | Temperature | Time | Cycles |
| Initial denaturation | 95°C | 3 min | 1 |
| Denaturation | 98°C | 20 sec | See table below |
| Annealing | 60°C | 15 sec | See table below |
| Extension | 72°C | 30 sec | See table below |
| Final extension | 72°C | 5 min | 1 |
| Hold | 4°C | Hold | - |
| Input DNA | Cycles for 100 ng library | Cycles for 1 μg library |
| 100 pg | 13–15 | 16–18 |
| 1 ng | 9–11 | 13–15 |
| 10 ng | 4–6 | 8–10 |
| 50 ng | 2–4 | 5–7 |
| 100 ng | 0–2 | 4–5 |
| 250 ng | / | 3–4 |
| 500 ng | / | 2–3 |
| 1 μg | / | 0–2 |
Cycle numbers are 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 additional cycles.
- Clean up with 45 μl beads (0.9×) per 50 μl reaction as above (two 200 μl 80% ethanol washes, air-dry 5–10 min). Elute in 25 μl eluent or ddH2O (ddH2O if targeted capture follows) and recover 20 μl. Safe stopping point: store at -20°C.
- Library quality control
- Check size distribution on an electrophoresis-based instrument (e.g. Bioanalyzer, TapeStation, LabChip GX or Fragment Analyzer) and concentration with a dsDNA fluorescent dye assay such as the GenieQuant 1× dsDNA HS Assay Kit.
Optional two-round size selection
Perform size selection only once. With ≥100 ng input, select after adapter ligation; with <100 ng or limited sample, select after amplification. Bring the sample to 100 μl with ddH2O, add X μl beads, incubate 5 min, magnetise and transfer the supernatant to a new tube (discard beads). Add Y μl beads, incubate 5 min, magnetise and discard the supernatant. Wash twice with 200 μl 80% ethanol, air-dry 3–5 min, elute in 25 μl and recover 20 μl.
| Expected insert size (bp) | 150 | 200 | 250 | 300 | 350 | 400 | 450 | 500 |
| After ligation, 1st round X (μl) | 78 | 68 | 65 | 59 | 56 | 53 | 51 | 50 |
| After ligation, 2nd round Y (μl) | 20 | 20 | 15 | 15 | 12 | 12 | 10 | 10 |
| After amplification, 1st round X (μl) | 78 | 70 | 63 | 55 | 50 | 46 | 45 | 44 |
| After amplification, 2nd round Y (μl) | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 15 |
| Stubby adapters, after ligation, 1st round X (μl) | 100 | 90 | 75 | 65 | 60 | 55 | 53 | 50 |
| Stubby adapters, after ligation, 2nd round Y (μl) | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 18 |
For stubby adapters after amplification, use the standard after-amplification conditions. Bead selection is ineffective for inserts >700 bp; use gel extraction instead. Do not size-select samples <50 μl directly.
Q1: How do I control library insert size?
- Adjust the 37°C fragmentation time: 25–30 min for 150 bp, 17–22 min for 250 bp, 10–15 min for 350 bp and 5–10 min for 550 bp. Fine-tune by 2–5 min if needed, and use two-round bead size selection for a tighter distribution.
Q2: My DNA is in TE buffer. Can I still use it?
- Yes, but the fragmentation enzyme is EDTA-sensitive. Either purify the DNA with 2.2× beads and elute in ddH2O, or add Neutralization Buffer according to the final EDTA concentration (e.g. 1 μl for 0.2 mM; none below 0.1 mM).
Q3: Can I make PCR-free libraries?
- Yes. With full-length adapters and sufficient input (≥50 ng without size selection, ≥200 ng with size selection), the library is complete after the ligation clean-up. Stubby adapters always require amplification.
Q4: How can I reduce adapter dimers?
- Use the recommended adapter concentration for your input, avoid unnecessarily long ligation times, and if dimers remain, perform an extra 1× bead clean-up (product made up to 50 μl, plus 50 μl beads).
Q5: Why does my library show a high-molecular-weight tail?
- This is usually over-amplification, where strands cross-anneal once primers are depleted. Reduce the cycle number rather than size-selecting. These libraries sequence normally, but use qPCR-based quantification as dsDNA dyes underestimate them.
Q6: Is the kit suitable for FFPE DNA?
- Yes. Use ≥50 ng FFPE DNA. The kit reduces artificial chimeric reads in FFPE libraries, and fragmentation time does not need adjusting for FFPE integrity unless insert size is critical.
