Sequencing While Synthesizing

the Core Principle of NGS

NGS is the process of randomly fragmenting and splicing DNA to prepare a sequencing library. By performing extension reactions on the tens of thousands of clones in the library, the corresponding signals are detected, and sequence information is finally obtained.

The core principle of NGS is sequencing-while-synthesizing, as analyzed in the following.

Cycle 1: Add reaction system

Incorporate a base

Remove other unincorporated nucleotides

Signal detection

Cycle 2 - n: Add reaction system, Repeat Cycle 1

11. Four fluorescently labeled NTPs are added to each round of sequencing reactions.

terminated with a protecting group that can be removed.

2. Only one nucleotide is integrated in each reaction round and the instrument reads the corresponding fluorescence signal.

3. End of signal reading, chemical removal of blocking and fluorescent groups for next round of sequencing reactions.

Currently, Illumina is one of the most widely used company in the market, and the prepared library is attached to a special chip “Flowcell” by the principle of base complementary pairing, in the following steps:

I. Flowcell 8 lanes, inner surface → chemically modified → 2 DNA primers → complementary to the splice sequence of the DNA library to be sequenced → covalently bonded to the Flowcell to prevent it from being washed away by the liquid

II. DNA library: many DNA fragments, joined at both ends with a specific DNA junction to form a DNA mixture → two features, production method → ultrasound to interrupt the genomic DNA, both ends with enzymes to fill, using Klenow enzyme to add an A base at the 3' end, and then ligase to join the junction → form a library → DNA mixture

III. Bridge PCR: seed the library onto the chip → complementary hybridization (DNA sequences at both ends of the library are complementary to the primers on the chip) → add dNTPs and enzymes → generate a new strand → add NaOH alkali solution → DNA strand unstranded → leave the original strand → add neutral liquid to neutralize the alkali solution → the other end of the DNA is complementarily hybridized with the second primer on the glass plate → add enzymes and dNTPs → add alkali → add neutral liquid → repeat the process for amplification

IV. Transform the synthesized double chain into a single chain that can be sequenced → chemical reaction → cut off a specific group on a primer → alkali solution washes the remaining chain of the chip → add neutral solution with sequencing primer (with fluorescence labeled dNTP → 3' end is blocked by an azide group → a cycle can only extend a base, polymerase → select the dNTP complementary to the base in the original position) →remove the excess dNTP and enzymes by water → put the base on the microscope for laser scanning → determine the base type (4 dNTPs) according to the fluorescence emitted → add chemical reagents to cut off the azide group and the fluorescent group labeled next to it → expose the hydroxyl group at the 3' end → add new dNTPs and new enzymes → extend one base again → flush out the excess enzymes and dNTPs → read the color in the laser scan → repeat the process

V. Index: Marking on the junction of the library; the specific sequence on the sample-specific junction marks the origin of the sample

VI. Read index: alkali unstranded read1 DNA → add neutral solution → add read2 sequencing primer (binding site next to index sequence) → perform 2 rounds of sequencing (generally 6 to 8 bases) → understand a specific segment of DNA from which sample of the original

VII. Double-end sequencing: one DNA strand is read once in both forward and reverse directions, doubling the effective length of sequencing

VIII. Inverted strand: synthesize DNA complementary strand → cut the root of the original strand with chemical reagents → remaining complementary strand → perform second sequencing (add read3 primers to read bases)