DNA Library Prep | Animal breeding samples (cattle, sheep, geese, chic

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	12330ES	Hieff NGS^TM Stubby UDI Primer Kit for Illumina, Set4（1,152 Illumina Dual-Index Unique Adapters, Plate-Format, Set 4）
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
Type	Cattle Tissue		Sheep Tissue		Chicken Tissue	Goose Tissue

Product description

Library Preparation Method	Automated library preparation: MGI SP-960
Input DNA	300 ng
Fragmentation	4℃ 1 min, 35℃ 15 min, 72℃ 20 min, 4℃ hold
Adapter	Illumina UDI adapter, used undiluted
Post-Ligation Cleanup & Size Selection	After adding 40 μL H₂O, proceed directly to size selection without purification, using a 0.4×/0.1× double-sided bead ratio.
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	30℃	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

Adapter-ligated products were brought to 150 μL with 40 μL nuclease-free H₂O and subjected directly to 0.4×/0.1× double-sided size selection. 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
Sample Type	Cattle		Sheep		Chicken	Goose
Input Amount	300 ng
Library Concentration (ng/μL)	42.4	44.4	40.8	41	53.6	49.4	50.2
Total Amount ng	1060	1110	1020	1025	1340	1235	1255
Qsep Main Peak(bp)	482	486	494	488	499	498	492

Library size distribution

Conclusion

Genomic DNA from animal breeding samples—cattle, sheep, goose, and chicken tissues—was used to construct libraries with the enzymatic fragmentation kit (Cat. No. 12972ES). The resulting libraries showed high yield, tight fragment size distribution, and met all QC criteria.

By adopting a purification-free, direct size selection workflow, the protocol saved ~30 minutes of hands-on time while delivering libraries with a sharp main peak and no primer dimer contamination.

DNA Library Prep | Animal breeding samples (cattle, sheep, geese, chickens) | WGS