Automated DNA Library Prep | Metagenomic Sequencing-Fecal DNA Samples

Metagenomic sequencing has become a core tool for untangling complex microbial ecosystems by enabling culture-independent, high-throughput analysis of all microbial genetic material in environmental and biological samples. Fecal samples are the most common research matrix for gut microbiome and microbial ecology studies, yet traditional manual library preparation suffers from low efficiency, high human error, and poor batch consistency. To solve these pain points, we established a standardized, automated fecal DNA library construction workflow based on the MGISP-960 high-throughput automated sample preparation platform (MGI), delivering stable, high-quality sequencing libraries for large-scale metagenomic research.

• Human microbiome & host health: obesity, diabetes, IBD, autism; probiotics and prebiotics.
• Clinical infectious disease diagnosis (mNGS): unknown pathogens plus resistant and virulence genes.
• Natural-environment microbial ecology: soil, marine, freshwater, and glacial systems.
• Modern agriculture & livestock: rhizosphere, biocontrol, and feed optimization.
• Industrial microbial resources: novel enzymes and fermentation optimization.
• Public health & epidemic control: port surveillance and emerging-pathogen tracing.

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	12330ES	Hieff NGS™ Stubby UDI Primer Kit for Illumina, Set4（1,152 Illumina Dual-Index Unique Adapters, Plate-Format, Set 4）
User-Supplied Materials	—	Absolute Ethanol

The Automated Workflow

Pre-Experiment Preparation

1. Equilibrate magnetic beads to room temperature before use.

2. Prepare 80% ethanol.

3. Prepare 2 herbal medicine samples and 1 rapeseed sample, each containing 400 ng of DNA. The extracted DNA shows visible pigment contamination.

Product description

Procedure

1. DNA Fragmentation / End Repair / dA-Tailing

Thaw all reagents listed in Table 1, invert to mix thoroughly, and keep on ice. On ice, prepare the reaction mixture as specified in Table 1. Gently pipette up and down or use low-speed vortexing to mix, then briefly centrifuge to collect the reaction liquid at the bottom of the tube. Perform DNA fragmentation, end repair, and dA-tailing according to the thermal cycling program in the table.

Table 1. PCR Reaction for DNA Fragmentation / End Repair / dA-Tailing

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Input DNA	100 ng	Heated Lid: 105 °C	On
Smearase™ Buffer 4.0	10	4℃	1 min
Smearase™ Enzyme 4.0	10	30℃	15 min
ddH2O	Up to 60	72℃	20 min
-	-	4℃	Hold

2. Adapter Ligation
The adapter should be diluted to an appropriate concentration based on the input DNA amount. In this experiment, the UDI (Unique Dual Index) adapters were used undiluted.

Thaw all reagents listed in Table 2, invert to mix thoroughly, and keep on ice. On ice, prepare the reaction mixture as specified in Table 2. Gently pipette up and down or vortex briefly to mix, then briefly centrifuge to collect the reaction liquid at the bottom of the tube. Perform the adapter ligation reaction according to the program in Table 2.

Table 2. Adapter Ligation Reaction

Name	Volume (μL)	Temperature	Time
dA-tailed DNA	60	-	-
Ligation Enhancer 4.0	30*	Heated Lid	Off
Rapid DNA Ligase 4.0	10	20℃	15 min
PE Adapter	5	4℃	Hold
ddH2O	Up to 110	-	-

【Note】:* Ligation Enhancer is viscous. Before use, invert and vortex thoroughly to mix completely, then briefly centrifuge.

3. Post Ligation Clean Up

This step uses magnetic beads to purify the adapter-ligated products. Purification removes unligated adapters or adapter dimers and other ineffective byproducts.

In this experiment, a post-ligation cleanup followed by size selection was used: perform 0.8× cleanup on the ligation product, elute with 102 μL ddH₂O, then carry out size selection at a ratio of 0.58×/0.15×. The final product was eluted in 20 μL for the next amplification step.

4. Library Amplification

This step performs PCR amplification to enrich the purified and size-selected adapter-ligated products. Prepare the reaction mixture and set the cycling program according to Table 3.

Table 3. Library Amplification Reaction

5. Magnetic Bead Purification of Amplified Products
The amplified products were purified using Hieff NGS™ DNA Selection Beads (0.45×, Beads:DNA = 0.45:1).

6. Library Quality Control

Sample Type	Fecal DNA 1	Fecal DNA 2	Fecal DNA 3	Fecal DNA 4	Fecal DNA 5
Library concentration (ng/μL)	71.8	68.4	61.8	70.8	49.6
Yield (ng)	2472	2460	2346

Agarose gel electrophoresis image of the libraries

Analysis of Experimental Results

Fecal 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.

Yeasen Product Portfolio for Metagenomic sequencing