How Many Functional Sequences Typically Comprise the Adapter of a Next-Generation Sequencing (NGS) Library
The adapter design of a Next-Generation Sequencing (NGS) library plays a critical role in enabling efficient and accurate sequencing. Typically, an adapter consists of multiple functional sequence elements that facilitate interactions with sequencing platforms, enable nucleic acid amplification, and support sample identification.
The following are common functional components found in NGS adapters:
1. Primer Binding Sites
These sequences enable the binding of sequencing primers or PCR primers during the sequencing workflow, allowing library fragments to be either amplified or directly sequenced.
2. Diversity Sequences
Some library preparation protocols incorporate one or more random nucleotides within the adapter to enhance sequence diversity. This promotes optimal cluster formation and detection on the sequencing platform.
3. Library Identifiers or Barcodes (Also Referred to as Indices)
These short nucleotide sequences allow for the multiplexing of different samples within a single sequencing run. After sequencing, reads can be demultiplexed based on these unique index sequences.
4. Platform-Specific Sequences
Certain sequencing platforms require proprietary adapter sequences to ensure compatibility with the instrument's chemistry and signal reading mechanisms.
5. P5 and P7 Binding Regions
For Illumina sequencing platforms, these sequences are necessary for immobilizing library fragments onto the flow cell surface during cluster generation.
6. Sequences for Melting Temperature Control
Specific sequences may be included to adjust the melting temperature of the adapter region, thereby optimizing the efficiency and specificity of PCR and other enzymatic reactions.
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