Automated DNA Library Prep | Whole‑Genome Re‑sequencing of Rice Leaf

This application note presents a fully automated DNA library-construction method for rice leaf samples on the MGISP-960 high-throughput automated sample preparation system, built on the Hieff NGS™ OnePot Pro DNA Library Prep Kit V4 (12972ES). Standardized, programmed operation eliminates manual error, improves library stability and throughput, and delivers sequencing-ready libraries of ~450 bp for downstream SNP / InDel detection, population genetics and molecular breeding.

Background and Pain Points

As rice breeding moves toward high-density genotyping of hundreds or thousands of accessions, library construction has become the throughput bottleneck of the re-sequencing workflow. The pain points of the manual bench workflow are:

•  Operationally intensive: fragmentation, ligation, bead cleanups and PCR setup require dozens of pipetting steps per sample.

•  Operator-dependent error: volume deviation, uneven bead-adsorption time and incomplete mixing shift insert-size distributions and yields.

•  Significant batch variation: libraries prepared on different days or by different operators are hard to compare across a breeding population.

•  Throughput ceiling: manual preparation cannot cost-effectively support large SNP / InDel panels and pan-genome projects.

Application Scenarios

The same automated backbone serves the full rice genomics portfolio — from variant discovery to breeding decision support.

 Figure 1. Application landscape covered by the automated rice leaf DNA library-construction workflow.

The Automated Workflow

The complete protocol runs on the MGISP-960 with the OnePot Pro DNA Library Prep Kit V4 (12972ES) and MGI-compatible UDB adapters. Manual touchpoints are limited to sample loading, adapter dilution (10 μM → 5 μM) and final library collection; all other settings follow the 12972ES instructions for use.

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12972ES	Hieff NGS™ OnePot Pro DNA Library Prep Kit V4
Magnetic Beads	12601ES	Hieff NGS™ DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	13350ES	Hieff NGS™ Unique Dual Barcode Primer Kit for MGI, Set 1 (plate format)
Automation Consumables	83560ES / 83672ES / 83130ES	0.2 mL 96-well PCR plates (white, full skirt) / 96-deep-well plates (U-bottom, 1.3 mL) / 250 μL filtered sterile tips
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 to a fully homogeneous suspension before deck loading.

•  Prepare fresh 80% ethanol for all bead washes.

•  Dilute the UDB adapter stock from 10 μM to 5 μM (manual pre-step, keep on ice).

•  Load one 96-sample consumable set (PCR plates, deep-well plates, filtered tips) and confirm deck layout, liquid volumes and tip integrity.

•  Verify input DNA quality (high-molecular-weight, free of visible pigment contamination) before the run.

Experimental Tips

Sample & input

•  Rice leaves accumulate polysaccharides and pigments that suppress bead binding — use cleaned, high-molecular-weight gDNA and keep the input at 200 ng consistently across the plate.

•  Assess gDNA integrity before automation; fragmented input lowers library yield and complicates insert-size control.

Bead-based purification & size selection

•  In dual size selection, the first bead ratio sets the lower cut-off and the second the upper cut-off: 0.62×/0.15× reproducibly delivers ~450 bp inserts for rice DNA.

•  Avoid over-drying beads after ethanol washes — 5–8 minutes of air-drying is enough; visibly cracked beads reduce elution recovery.

Adapter handling & PCR

•  Dilute adapter stocks manually before the run and keep them on ice; unique dual barcode adapters prevent index hopping.

•  6 PCR cycles are sufficient for 200 ng input and keep the duplication rate low.

Automation & QC

•  Run scripts exactly as specified in the 12972ES IFU — plate geometry and liquid classes are validated together.

•  Accept finished libraries at 30–45 ng/μL (Qubit) with a sharp ~450 bp peak (Qsep) before pooling.

Experimental Conditions and Results

Case results (Qubit 4.0):

All eight libraries fell between 30 and 45 ng/μL with a total yield of 845–1,075 ng; Qsep spot-checks showed insert sizes of approximately 450 bp, meeting sequencing-ready specifications.

Library Size Distribution

Figure 4. Rice leaf DNA library size distribution with a sharp single peak at ~450 bp.

Analysis of Experimental Results

Rice leaf samples were constructed into libraries using the enzymatic fragmentation library preparation kit 12972ES. The library yield was high, and the library fragment size distribution was concentrated, meeting quality control standards.