DNA Library Prep | Inhibitor-Rich Environmental Samples (Soil, feces,

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12972ES	Hieff NGS^TM OnePot Pro DNA Library Prep Kit V4
Magnetic Beads	12601ES	Hieff NGS^TM DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	13350ES	Hieff NGS^TM Unique Dual Barcode Primer Kit for MGI^TM, Set1
User-Supplied Materials	—	Absolute Ethanol

Pre-Experiment Preparation

1. Allow the magnetic beads to equilibrate to room temperature before use.

2. Prepare 80% ethanol.

3. Dilute the adapter 2-fold.

4. Prepare the sample according to the quantities listed in the table below.

No.	1	2	3	4	5	6	7	8
Sample Type	Pig feces		Mouse feces		Soil		Enteric contents

Product description

Library Preparation Method	Automated library preparation: MGI SP-960
Input DNA	200 ng
Fragmentation	4℃ 1 min, 35℃ 15 min, 72℃ 20 min, 4℃ hold
Adapter	Illumina UDI adapter, 2-fold dilution
Post-Ligation Cleanup & Size Selection	0.7× purification after ligation, Size selection 0.62×/0.15×
PCR Cycles	6 cycles
Post-PCR Cleanup	0.9× purification
Library Elution Volume	25 μL

Procedure

1. DNA Fragmentation / End Repair / dA-Tailing

Thaw and invert-mix all reagents in Table 1; keep on ice. Prepare the reaction mix on ice, mix gently (pipette or low-speed vortex), then spin briefly. Run the thermal program for fragmentation, end repair, and dA-tailing.

Table 1.DNA Fragmentation / End Repair / dA-Tailing

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Input DNA	X	Heated Lid: 105 °C	On
Smearase Buffer 4.0	10	4℃	1 min
Smearase Enzyme 4.0	10	35℃	15 min
ddH2O	Up to 60	72℃	20 min
-	-	4℃	Hold

2. Adapter Ligation
Adjust the Adapter concentration appropriately based on the Input DNA amount. For this experiment, dilute the UDI adapters 2-fold.

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 adapter-ligated products, removing unligated adapters and adapter dimers.

A “purify-then-size-select” strategy was used: first, 0.7× cleanup followed by elution in 102 μL ddH₂O; then, double-sided size selection at 0.62×/0.15×. The final product was eluted in 20 μL for downstream amplification.

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

Name	Volume (μL)	Temperature	Time	Cycle Numbe
Adapter Ligated DNA	20	98℃	45 sec	1
2×Ultima HF Amplification Mix	25	98℃	15 sec	6
Primer Mix(12330ES)	5*	60℃	30 sec
Total	50	72℃	30 sec
-	-	72℃	1 min	1
-	-	4℃	Hold	-

5. Magnetic Bead Purification of Amplified Products
The product after amplification is purified using Hieff NGS^TM DNA Selection Beads (0.9×, Beads:DNA = 0.9:1).

Library Quality control results

No.	1	2	3	4	5	6	7	8
Sample Type	Pig feces		Mouse feces		Soil		Enteric contents
Input Amount	32.2	37.4	36.2	42.6	39.4	46.4	29.4	35.2
Library Concentration (ng/μL)	805	935	905	1065	985	1160	735	880
Library Peak	421 bp	435 bp	453 bp	449 bp	452 bp	445 bp	421 bp	448 bp

Library size distribution(Qseq Ananlysis)

Conclusion

For samples with high levels of humic acid and other impurities, library preparation was performed using the enzymatic DNA fragmentation kit (Cat. No. 129772ES) on the MGISP-960 automated workstation. The resulting libraries showed excellent uniformity in yield and a tight main peak between 400–450 bp, meeting all QC requirements.

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DNA Library Prep | Inhibitor-Rich Environmental Samples (Soil, feces, and enteric samples) | WGS