DNA Library Prep | Aquatic animals | WGS

Reagent List for the Experiment

Category	Cat.No.	Product name
DNA Library Preparation	12972ES	Hieff NGSTM OnePot Pro DNA Library Prep Kit V4
Magnetic Beads	12601ES	Hieff NGSTM DNA selection Beads (Superior Ampure XP alternative)
Quantification	12642ES	1× dsDNA HS Assay Kit dsDNA qubit
Adapters	12330ES	Hieff NGSTM 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. Equilibrate magnetic beads to room temperature before use.

2. Prepare 80% ethanol.

3. Dilute the adapters 2-fold.

4. Prepare the samples according to the volumes specified in the table below.

Product description

Procedure

1. DNA Fragmentation / End Repair / dA-Tailing

After thawing the reagents listed in Table 1, vortex to mix, and place on ice for later use. Prepare the reaction mixture (as shown in Table 1) on ice. Mix gently by pipetting or vortexing at low speed, then perform a quick pulse centrifugation to collect the reaction solution at the bottom of the tube. Incubate according to the reaction program in the table to perform DNA fragmentation, end repair, and dA-tailing reactions.

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

Reaction System		Reaction Program
Reaction Component	Volume (μL)	Temperature	Time
Input DNA	200 ng	Heated Lid: 105 °C	On
SmearaseTM Buffer 4.0	10	4℃	1 min
SmearaseTM Enzyme 4.0	10	35℃	15 min
ddH2O	Up to 60	72℃	20 min
-	-	4℃	Hold

2. Adapter Ligation
The Adapter concentration must be diluted according to the Input DNA amount. For this experiment, the UDI adapters are diluted 2-fold.

After thawing the reagents listed in Table 2, vortex to mix and place on ice for later use. Prepare the reaction mixture (as shown in Table 1) on ice. Mix gently by pipetting or vortexing, then perform a quick pulse centrifugation to collect the reaction solution at the bottom of the tube. Incubate according to the program in Table 2 to perform the adapter ligation reaction.

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
UDB 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, 0.6× cleanup followed by elution in 102 μL ddH₂O; then, double-sided size selection at 0.65×/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

5. Magnetic Bead Purification of Amplified Products
The amplified products were purified using Hieff NGS^TM DNA Selection Beads(0.9×，Beads:DNA=0.9:1)

6. Library Quality Control

 

Library Size Distribution

a. Agarose Gel Electrophoresis of the Libraries

[Note]: The brightest band of the Marker indicates 500 bp)

b. Qsep analysis (samples 1–4)

Analysis of Experimental Results

Aquatic samples—including swamp eel, oyster, scallop, and crayfish—were processed using the enzymatic fragmentation library prep kit (Cat. No. 12972ES) on the AMTK iLab300 platform. The resulting libraries exhibited high yield and tight fragment size distribution, meeting all QC requirements.