In the era of rapid technological advancement, Next-Generation Sequencing (NGS) library preparation technology, serving as the decoder of the DNA world, plays an increasingly critical role. It has demonstrated significant value in areas such as cancer gene detection, genetic disease screening, and microbiome research, driving revolutionary changes in scientific research and clinical medicine.
The NGS workflow broadly encompasses: nucleic acid extraction → library preparation → target capture → sequencing → bioinformatic analysis. The core of library preparation technology lies in transforming target DNA/RNA molecules into libraries compatible with high-throughput sequencing instruments, thereby acquiring sequence data containing vital biological information.
Picture 1: NGS workflow
Using DNA library construction as an example. The workflow includes: DNA Fragmentation → End Repair and A-tailing → Adapter Ligation → Purification and Sorting → Library Amplification → Library Purification.
Picture 2: DNA library construction workflow
DNA Fragmentation
Due to the limited read lengths of current NGS platforms, extracted DNA samples require fragmentation. This is typically achieved via enzymatic or mechanical methods.
- Mechanical Fragmentation: Produces DNA fragments with a tight size distribution and minimal sequence bias, considered the gold standard for fragmentation in library prep, albeit at a slightly higher cost.
- Enzymatic Fragmentation: Utilizes sequence-agnostic fragmentation enzymes, offering stable fragmentation results without the need for complex mechanical instrumentation. The size of the resulting fragments depends solely on enzymatic digestion time.
It is noteworthy that while various fragmentation enzymes exist, most operate on endonuclease principles. Among these, Tn5 transposase remains dominant in the NGS market for its ultra-fast library construction capabilities, although its inherent sequence bias limits its application scope. Enzymes like DNase I, Endonuclease V, and Fragmentase (a blend of enzymes) show promise but face limitations preventing their widespread adoption. Fragmentase is currently the most widely recognized enzymatic fragmentation method for NGS.
Table 1: Principles and Limitations of Various Fragmentation Enzymes
|
Enzyme Class |
Dnase 1 |
Endonuclease V |
Fragmentase |
Tn5 |
|
Composition |
Single enzyme |
Single enzyme |
Enzyme mixture |
Single enzyme |
|
Type |
Endonuclease |
Endonuclease |
Endonuclease |
Transposase |
|
Principle |
Digests DNA randomly into fragments of desired size under different cation conditions. |
Controls DNA digestion length by adjusting uracil content in DNA. |
One enzyme nicks DNA; the other recognizes nicks and cleaves the complementary strand, fragmenting DNA to desired size. |
Generates random DNA fragments via transposon-mediated insertion at specific sites. |
|
limitation |
Experiments are influenced by multiple factors, resulting in poor reproducibility. Also exhibits sequence bias in cleavage sites. |
GC content of samples potentially affects fragmentation efficiency due to sequence composition differences. Practical operation is also susceptible to multiple factors. |
/ |
Exhibits sequence bias in the 9 base pairs flanking the insertion sites. |
End Repair and A-tailing
Fragmented DNA may possess 5'/3' overhangs or blunt ends. All overhangs must be converted to blunt ends: 3' overhangs are trimmed and 5' overhangs are filled in. For subsequent adapter ligation using the TA cloning strategy, DNA fragments require 5' phosphorylation and the addition of a single 'A' nucleotide to the 3' ends. This creates compatible ends for ligation to adapters featuring a single 3' 'T' overhang. These enzymatic steps are performed collaboratively by T4 DNA Polymerase, T4 Polynucleotide Kinase，and Taq DNA Polymerase.
Adapter Ligation
Adapters are crucial components of sequencing libraries. Taking the commonly used Y-shaped Illumina adapters as an example, they contain the P5/P7 flow cell binding sequences, Index sequences for sample multiplexing, and the Rd1/Rd2 SP binding sites.
- The P5/P7 sequences hybridize to complementary oligonucleotides on the flow cell, immobilizing fragments for bridge amplification.
- Indexes allow differentiation of samples within pooled libraries during sequencing. Ligation is typically catalyzed by T4 DNA Ligase, which seals nicks in double-stranded DNA, joining the adapter (with its 3' 'T' overhang) to the A-tailed DNA fragment, forming a complete double-stranded molecule.
Adapter design has diversified with sequencing technology advancements, including single-end/paired-end adapters, UMI adapters, transposase-compatible adapters, and full-length/short adapters, catering to diverse applications. Adapters can be categorized by:
- Platform: Illumina adapters, MGI adapters.
- Type:Full-length (long) adapters, stubby (short) adapters.
- Indexing Strategy:CDI (Combinatorial Dual Index) adapters, UDI (Unique Dual Index) adapters.
Picture 3: Single-Indexed Library Construction on Illumina and MGI Platforms
Picture 4: Single-Indexed Library Construction on Illumina and MGI Platforms
Purification and Size Selection
This step typically involves a two-stage magnetic bead-based purification. Leveraging the principle that beads preferentially bind larger DNA fragments, different bead-to-sample volume ratios are used:
- First Purification:Removes large fragments and undesired complexes .
- Second Purification:Removes small fragments and residual reagents.
This isolates the library within the desired fragment size range.
Picture 5: DNA fragment purification
Picture 6: DNA Fragment size selection
Library Amplification
Polymerase Chain Reaction (PCR) is employed to generate sufficient quantities of adapter-ligated DNA fragments for sequencing. PCR typically utilizes high-fidelity DNA polymerase. This enzyme possesses 5'→3' polymerase activity for DNA synthesis and 3'→5' exonuclease activity, enabling it to correct misincorporated nucleotides during amplification. This facilitates rapid and high-fidelity amplification of the library fragments.
Guideline for NGS core enzymes in DNA & RNA library construction
Yeasen is a biotechnology company engaged in the research, development, production, and sales of three major biological reagents: molecules, proteins, and cells. Yeasen Biotech company produces a variety of enzymes related to NGS library construction. You can choose the most suitable library construction product from the chart below.
Table 1. Guideline for NGS core enzymes in DNA & RNA library construction
|
Type |
Product positioning |
Product name |
Cat# |
|
RNA library construction |
rRNA depletion/2nd strand cDNA synthesis |
12906ES |
|
|
rRNA depletion |
10325ES |
||
|
1st strand cDNA synthesis |
14672ES |
||
|
11112ES |
|||
|
2nd strand cDNA synthesis |
12903ES |
||
|
RNA library construction & DNA library construction |
End repair |
12901ES |
|
|
12902ES |
|||
|
dA-Tailing |
13486ES |
||
|
Adapter ligation |
10301ES |
||
|
PCR amplification |
2×Super Canace™ II High-Fidelity Mix for Library Amplification |
12621ES |
Table2. DNA & RNA Library Prep Kit
|
|
Name |
Cat# |
Notes |
|
DNA |
Hieff NGS DNA Library Prep Kit |
13577ES |
Tumor/ Mechanic method |
|
Hieff NGS OnePot Pro DNA Library Prep Kit V2 |
12194ES |
Tumor/ Enzymetic method |
|
|
Hieff NGS OnePot II DNA Library Prep Kit for Illumina |
13490ES |
Pathgen/ Enzymetic/ regular time (140min) |
|
|
Hieff NGS OnePot Flash DNA Library Prep Kit |
12316ES |
Pathgen/ Enzymetic/ Ultrafast (100min) |
|
|
12305ES |
Pathgen/ Enzymetic/ DNA & RNA Co-Prep |
||
|
RNA |
12308ES |
Without oligo dT magnetic beads, 11 tubes |
|
|
12309ES |
oligo dT magnetic beads plus, 14 tubes |
||
|
Hieff NGS™ Ultima Dual-mode RNA Library Prep Kit |
12310ES |
Premixed version, 5 tubes |
|
|
Hieff NGS™ EvoMax RNA Library Prep Kit（Premixed version）(actinomycin D Free) |
12340ES |
Premixed version, (Actinomycin D Free) |
|
|
12254ES |
Plant |
||
|
12257ES |
Human |