Soybean is a major oil and protein crop, and leaf-tissue re-sequencing underpins basic variant detection, population genetics, leaf-trait gene mining and molecular breeding. Germplasm panels span hundreds of accessions whose leaves — young or mature — yield DNA of variable quantity and quality, so the library workflow must tolerate low input while staying strictly consistent.
This application note establishes an automated whole-genome re-sequencing library-construction workflow for soybean leaf DNA on the MGISP-960 platform using the Hieff NGS™ OnePot Pro DNA Library Prep Kit V4 (12972ES). The workflow is validated at 10 ng and 25 ng inputs, delivers Illumina-compatible libraries, and gives breeding programs a standardized, high-throughput route from leaf tissue to sequencing-ready libraries.
Background and Pain Points
Soybean breeding programs push the library workflow to its limits in four ways:
• Panel scale: re-sequencing hundreds of accessions turns library construction into an industrial batch process.
• Variable input: leaf age and extraction batch cause DNA yields to swing — the workflow must perform at 10–25 ng.
• Turnaround Time: : breeding cycles leave little room for 6–8 hour manual sessions.
• Cross-season comparability: datasets collected across seasons and sites must be built with identical, traceable programs.
Application Scenarios
From variant calling to trait-gene mining, one automated backbone serves the whole soybean breeding pipeline.
Figure 1. Application landscape covered by the automated soybean leaf DNA library-construction workflow.
The Automated Workflow
The protocol runs on the MGISP-960 with the OnePot Pro DNA Library Prep Kit V4 (12972ES) and Illumina-compatible PE adapters. One 96-sample run consumes 8 deep-well plates, 6 PCR plates and 7 boxes of filtered tips.
Figure 2. Automated soybean leaf DNA library-preparation workflow on the MGISP-960 (Illumina library).
Reagent List for the Experiment
|
Category |
Cat.No. |
Product name |
|
DNA Library Preparation |
12972ES |
|
|
Magnetic Beads |
12601ES |
Hieff NGS™ DNA selection Beads (Superior Ampure XP alternative) |
|
Quantification |
12642ES |
|
|
Adapters |
12327–12330ES |
|
|
Automation Consumables |
83560ES / 83672ES / 83130ES |
0.2 mL 96-well PCR plates (white, full skirt) / |
|
Sealing |
83624ES |
qPCR pressure-sensitive sealing film (scraper included) |
|
User-Supplied Materials |
— |
Absolute Ethanol |
Pre-Experiment Preparation
• Equilibrate DNA Selection Beads to room temperature for at least 30 minutes and resuspend fully before deck loading.
• Prepare fresh 80% ethanol for all bead washes.
• Keep the PE adapter working solution at 2 μM (manual pre-step, keep on ice).
• Load 8 deep-well plates, 6 PCR plates and 7 boxes of filtered tips for one 96-sample run; confirm deck layout and liquid volumes.
• QC the leaf DNA input (concentration, purity and integrity) before the run; young leaves generally give cleaner, higher-yield DNA.
Experimental Tips
Sample & input
• The workflow is validated at 10 ng and 25 ng inputs; keep the chosen mass consistent across the plate.
• Harvest young, healthy leaves where possible — mature leaves carry more phenolic compounds that inhibit enzymatic steps.
Bead-based purification & size selection
• Post-ligation purification uses 0.6× beads (66 μL) eluted in 104 μL; size selection at 0.60×/0.15× follows, with 22 μL elution and 20 μL transferred to PCR.
• Avoid over-drying beads; cracked pellets reduce recovery, which is costly at 10 ng input.
Adapter handling & PCR
• PE adapter at 2 μM with 20 °C 15-minute ligation is the validated low-input setting.
• 11 PCR cycles with 5 μL index primer balance yield and duplication for 10–25 ng inputs.
Automation & QC
• Follow the 12972ES IFU scripts exactly; the low-input program (4 °C 1 min → 30 °C 12–15 min → 72 °C 20 min) is part of the validated package.
• Quantify with the 1× dsDNA HS Assay Kit and confirm a sharp single peak on the electropherogram before pooling.
Experimental Conditions and Results
|
Parameter |
Setting |
|
Platform |
MGISP-960 |
|
Sample type |
Plant material as indicated |
|
Input amount |
10 ng or 25 ng genomic DNA |
|
Library prep kit |
Hieff NGS™ OnePot Pro DNA Library Prep Kit V4 (12972ES) |
|
Library type |
Illumina |
|
Enzymatic fragmentation / end repair / dA-tailing |
4°C 1 min → 30°C 12–15 min → 72°C 20 min → 4°C hold |
|
Adapter ligation |
PE adapter 2 μM, 5 μL; 20°C 15 min, 4°C hold |
|
Post-ligation purification |
0.6× beads (66 μL), elute in 104 μL |
|
Size selection |
0.60× / 0.15×; elute in 22 μL, transfer 20 μL to PCR |
|
PCR amplification |
Index primer 5 μL, 11 cycles |
|
Post-PCR purification |
0.8× beads (40 μL), final library eluted in 32 μL |
|
Consumables per 96-sample run |
8 × deep-well plates, 6 × PCR plates, 7 × boxes filtered tips |
Validated QC results: 963 ng total yield from the 10 ng input and 1,926 ng from the 25 ng input — yield scales proportionally with input, and final libraries show a sharp single peak on the electropherogram, meeting sequencing-ready specifications.
Figure 3. Soybean leaf DNA library yield at 10 ng versus 25 ng input.
Analysis of Experimental Results
Libraries were constructed from DNA extracted from Soybean Leaf using the enzymatic fragmentation library‑prep kit 12972ES. High library yield and tight fragment‑size distribution were achieved, which meets standard quality‑control criteria.