Unpacking the Core Enzymes in NGS Library Workflow

Figure 1. Next-generation sequencing workflow^[2]

• DNA library preparation and its key enzymes
• RNA library preparation and its key enzymes
• Guideline for NGS core enzymes in DNA & RNA library Preparation

1. DNA Library Preparation and the Key Enzymes Involved

DNA library preparation is a critical step in next-generation sequencing (NGS), laying the groundwork for accurate and reproducible sequencing results. The objective is to convert input genomic DNA into a sequencing-ready format by fragmenting it, adding adapter sequences, and amplifying the final product. Each step relies on the precise action of one or more enzymes.

1.1 DNA Fragmentation

Fragmentation breaks the genomic DNA into appropriately sized pieces, typically between 150–500 bp depending on the sequencing platform.

Key enzymes:

• Dual-Enzyme Fragmentation Based on N* Patents: This system employs engineered enzymes such as mutant Vvn and mutant T7 Endonuclease I to fragment DNA in a sequence-independent manner.
• Non-Restriction Endonuclease-Based Random Cleavage: These approaches utilize nucleases like DNase I, Endonuclease V, or their engineered variants to randomly cleave DNA without sequence specificity.
• Transposase (in tagmentation-based methods): simultaneously fragments and tags DNA with adapter sequences (e.g., in Nextera-like workflows)

Yeasen’s OnePot DNA Fragmentation Enzyme series belongs to the second category. It features a proprietary mutant of a non-restriction endonuclease, offering efficient and uniform DNA fragmentation suitable for high-quality library preparation.

Figure 2. Base Composition Bias Analysis of Different DNA Fragmentation Methods

Comparison of base frequency distribution across sequencing reads generated using mechanical shearing, transposase-based fragmentation, and random endonuclease digestion. Transposase-based methods may exhibit positional bias due to sequence preference, while random endonucleases and mechanical shearing generally result in more uniform base composition profiles.

1.2 End Repair and A-Tailing

After DNA fragmentation, the resulting fragments often have uneven or incompatible ends. To prepare these for adapter ligation, two enzymatic modifications are typically performed: end repair and 3′ A-tailing (adenylation). In most commercial NGS library prep workflows, these two steps are combined into a single-tube reaction, streamlining the protocol and reducing sample loss.

The end repair process converts overhangs into blunt ends and introduces 5′ phosphate groups to enable ligation. Simultaneously, a single adenosine (A) is added to the 3′ ends of each fragment to facilitate ligation with T-tailed adapters.

Key enzymes involved include:

T4 DNA Polymerase(Cat#12901): fills in 5′ overhangs and removes 3′ overhangs

T4 Polynucleotide Kinase (PNK, Cat#12902): phosphorylates 5′ ends to enable ligation

Taq DNA Polymerase or Klenow Fragment (exo–): adds a single 3′ A-overhang; commonly used despite lower fidelity due to its terminal transferase activity

This enzymatic combination ensures that DNA fragments are blunt-ended, phosphorylated, and A-tailed, making them fully compatible with downstream adapter ligation steps.

Figure 3. Multiple enzymes are involved in the end repair process

Summary of Key Enzymes in DNA Library Prep:

Step	Enzyme Name	Function
Fragmentation	DNase I, Transposase	Randomly fragment DNA
End Repair	T4 DNA Polymerase, PNK, Klenow	Polish ends, phosphorylate for ligation
A-tailing	Taq Polymerase, Klenow (exo–)	Add A-overhangs to 3′ ends
Adapter Ligation	T4 DNA Ligase	Ligate adapters to DNA ends
PCR Amplification	High-fidelity polymerase (Q5, Phusion, etc.)	Amplify library with minimal errors

 2. RNA Library preparation and the Key Enzymes Involved

RNA library preparation for next-generation sequencing (NGS) is a multi-step process designed to convert RNA molecules into sequencing-ready DNA libraries. The specific steps vary depending on the RNA type (e.g., mRNA, total RNA, small RNA), but all protocols share common enzymatic requirements.

Comparison Table: RNA Types and Library Preparation Strategies

RNA Type	Library preparation Strategy	Key Enzymatic Steps Involved	Notes
mRNA	Poly(A) enrichment → cDNA synthesis → standard library	Reverse Transcriptase, DNA Polymerases, Ligase, HiFi Pol	Most common; high-quality input needed
Total RNA	rRNA depletion → cDNA synthesis → standard library	RNase H, Reverse Transcriptase, DNA Polymerases, etc.	Suitable for samples without poly(A); enables detection of ncRNA
miRNA / small RNA	Adapter ligation → Reverse transcription → PCR	T4 RNA Ligase, Reverse Transcriptase, HiFi Polymerase	Requires special adapters and gel selection for size discrimination
circRNA	rRNA depletion → RNase R treatment → cDNA synthesis	RNase H, RNase R, Reverse Transcriptase, etc.	Detects back-spliced junctions; no poly(A)
lncRNA	Same as mRNA or total RNA workflow	Reverse Transcriptase, DNA Polymerases, HiFi Polymerase	Enrichment depends on sample and research focus

 

Figure 4. RNA Library preparation workflow

The core steps typically include reverse transcription, second-strand synthesis, end repair/A-tailing, adapter ligation, and PCR amplification. When working with total RNA, additional steps such as rRNA depletion or poly(A) enrichment are required to enrich the target RNA population.

Below is an overview of the essential enzymes involved in RNA library preparation:

2.1 Reverse Transcription

The first step in RNA library prep is converting RNA into complementary DNA (cDNA).

Key enzyme:

Reverse Transcriptase (RT): Common examples: M-MLV RT, SuperScript II/III/IV, Hieff First-Strand cDNA Synthesis Enzyme Mix

Converts RNA into single-stranded cDNA with or without template switching

2.2 Second-Strand Synthesis and End Repair/A-Tailing (Combined Step)

The single-stranded cDNA generated during reverse transcription is inherently unstable, making immediate second-strand synthesis essential. This step is carried out using DNA Polymerase I, with the assistance of RNase H, which degrades the RNA portion of the RNA–DNA hybrid. Working in tandem, RNase H(Cat#12906) and DNA Polymerase I (Cat#12903) catalyze the synthesis of the complementary second strand, using the first-strand cDNA as a template. DNA Polymerase I exhibits 5′→3′ polymerase activity, enabling it to synthesize the second strand in a template-dependent manner.

The subsequent steps in the process include adapter ligation, and PCR amplification, all of which are detailed in the DNA library preparation procedure and need not be reiterated here. It's worth noting that once reverse transcription is completed, there's no need for further fragmentation of the nucleic acid fragment.

Summary of Key Enzymes in RNA Library Prep:

Step	Enzyme Name	Function
RNA Selection (Optional)	RNase H (for rRNA depletion) Oligo(dT) beads (for mRNA enrichment, no enzymes)	Enrich for mRNA (via poly(A) selection) or remove rRNA (via RNase H digestion)
First-Strand cDNA Synthesis	Reverse Transcriptase (e.g., M-MLV, SuperScript IV)	Reverse transcription of RNA into single-stranded cDNA
Second-Strand Synthesis + End Repair + A-Tailing (Combined Step)	RNase H, DNA Polymerase I, Taq Polymerase, Klenow (exo–)	Generates double-stranded cDNA and prepares ends for ligation
Adapter Ligation	T4 DNA Ligase	Ligation of sequencing platform-specific adapters to cDNA fragments
PCR Amplification	High-fidelity DNA Polymerases (e.g., Q5, Phusion, Hieff Canace™ HiFi)	Enrichment of adapter-ligated fragments to generate sequencing-ready library

 

3. Guideline for NGS core enzymes in DNA & RNA library preparation

Yeasen Biotech is a leading biotechnology company specializing in the research, development, manufacturing, and sales of core biological reagent systems across three major areas: molecular biology, protein science, and cell biology. As part of its molecular portfolio, Yeasen offers a comprehensive range of high-performance enzymes and kits designed for next-generation sequencing (NGS) library preparation. The chart below helps you select the most suitable library preparation solution based on your specific application needs.

 

Category		Name	Cat. No.
DNA Lib Prep	Mechanical	Hieff NGS™ DNA Library Prep Kit 2.0 (Mechanical)	12927ES
	Mechanical	Hieff NGS™ OnePot Pro DNA Library Prep Kit V4 (Enzymatic)	12972ES
		Hieff NGS™ OnePot Flash DNA Library Prep Kit (Enzymatic)	12316ES
	Fragmentase	Hieff NGS™ OnePot Pro DNA Fragmentation Module (end repair and dA-tailing)	12619ES
	End Repair and A-Tailing enzyme	T4 DNA Polymerase(5 U/μL)	12901ES
		T4 polynucleotide Kinase(10 U/μL)	12902ES
		S-Taq DNA polymerase	13486ES
RNA Lib Prep	Dual-mode(Strand specific & Non Strand specific)	Hieff NGS™ Ultima Dual-mode RNA Library Prep Kit	12308ES
	Premix version	Hieff NGS™ EvoMax RNA Library Prep Kit (Strand-specific)	12340ES
		Hieff NGS™ EvoMax RNA Library Prep Kit（dNTP）（Premix and Sealing Version）	12341ES
mRNA isolation	Eukaryotic mRNA	Hieff NGS™ mRNA Isolation Master Kit V2	12629ES
rRNA depletion	Human/Mouse/Rat	Hieff NGS™ MaxUp Human rRNA Depletion Kit ( Human/Mouse/Rat)	12257ES
	Human/Mouse/Rat	Hieff NGS™ MaxUp rRNA Depletion Kit (Plant)	12254ES
		Hieff NGS™ RNA Cleaner	12602ES

 

References：

[1] Mardis, Elaine R. Next-Generation Sequencing Platforms[J]. Annual Review of Analytical Chemistry, 2013, 6(1):287-303.
[2] Gulilat M, Lamb T, Teft W A, et al. Targeted next generation sequencing as a tool for recision medicine[J]. BMC Medical Genomics, 2019, 12(1):81.
[3] Lundberg K S, Dan D S, Adams M, et al. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus[J]. Gene, 1991, 108(1):1-6.
[4] Miyazaki K. Random DNA fragmentation with endonuclease V: application to DNA shuffling[J]. Nucleic Acids Research, 2002, 30(24):e139.
[5] Baldwin A, Morris A R, Mukherjee N. An Easy, Cost-Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA-seq[J]. Current Protocols, 2021, 1(6):e176.