The sequencing principle involves several key components, each playing a crucial role in the overall process. Here are the significant components and their functions:
1. DNA Nanoballs (DNBs):
These are formed during the library preparation and amplification process. DNBs consist of multiple copies of the same DNA fragment, which enhances the signal during sequencing by providing a higher concentration of the target DNA .
2. Reversible Terminator Nucleotides:
Initially used in the sequencing process, these nucleotides allow for the incorporation of a single base at a time. After each incorporation, the fluorescent signal is captured, and the terminator is removed to allow the next base to be added. This step is crucial for accurate base calling during sequencing .
3. MDA Polymerase:
This enzyme is used during the amplification phase after the first read (Read1) is completed. It replaces the reversible terminator nucleotides with regular dNTPs, allowing for the continuous extension of the DNA strand. This step is essential for generating sufficient DNA for the second read (Read2) and for the overall amplification of the library , .
4. Read1 and Read2:
These refer to the two sequencing reads that are generated. Read1 is the first sequence obtained, while Read2 is the complementary sequence obtained after the first read. The ability to read both strands of the DNA fragment increases the accuracy and reliability of the sequencing results .
5. Index Read:
After completing Read1 and Read2, an index read is performed. This step allows for the identification of different samples in multiplexed sequencing runs, enabling multiple samples to be sequenced simultaneously and efficiently .
Each of these components is integral to the sequencing workflow, ensuring that the process is efficient, accurate, and capable of generating high-quality genomic data.
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