How do I read the nucleotides in NGS?

In sanger sequencing, the nucleotides can be read in two main ways: through automated and manual methods. 

In automated Sanger Sequencing a computer reads each band of the capillary gel, utilizing fluorescence to identify each terminal ddNTP. As each fragment reaches the end of the tube, the laser illuminates it, allowing detection of the attached dye. With unique fluorescent labels for each ddNTP, the emitted light directly exhibits the terminal ddNTP's characteristics. The result is a chromatogram that displays fluorescent peaks for each nucleotide along the template DNA's length. 

In gel electrophoresis, short DNA fragments move through the gel pores rapidly, while longer fragments move more slowly. The smallest fragment (terminating just one nucleotide after the primer) crosses the end of the gel first, followed by progressively larger fragments. By analyzing the sequence of dye colors detected in succession, the original DNA sequence is formed nucleotide by nucleotide. The recorded data appear as a series of peaks in fluorescence intensity, from which the DNA sequence is analyzed. 

In manual Sanger Sequencing one should read all four lanes of the gel simultaneously (moving from bottom to top). The lane helps determine the terminal ddNTP for each band. 

In illumina, the sequencer captures images of the flow cell, recording the emission from each cluster. By analyzing the fluorescent emission intensity and wavelength, the sequence of the templates is identified. The fluorescent signals are converted into base calls (A, T, G, or C). This process involves identifying the sequence of each cluster based on the emitted light. After base calling, the generated reads are aligned to a reference genome. This step involves mapping the reads to their corresponding positions on the reference genome, allowing for the identification of the location of each sequenced fragment.