Next-Gen Sequencing

Next-generation sequencing (NGS), aka sequencing by synthesis, represents a suite of advanced technologies that allow for the rapid sequencing of DNA and RNA much faster and at a lower cost than traditional methods like Sanger sequencing. These technologies have revolutionized genomics, enabling large-scale studies and applications that were previously impossible or impractical.

NGS allows for simultaneous sequencing of the entire genome in hundreds of thousands of extremely small wells.

Process:

Library Preparation (refers to the collection of fragments):

• Fragment DNA or RNA into smaller pieces

• Attach adapters to the ends of these fragments (adapters help primers bind and index sequences)

• Amplify the fragments to create a library

  Cluster Generation:

• Load the DNA library onto a flow cell

• Amplify the fragments on the flow cell to form clusters of identical sequences

  Sequencing:

• Use sequencing by synthesis to add fluorescently labeled nucleotides one at a time

• Capture the emitted fluorescence to determine the sequence of each fragment

Sequencing by Synthesis:

Pyrosequencing is a powerful sequencing-by-synthesis method that relies on detecting light signals generated by the enzymatic conversion of pyrophosphate to ATP during nucleotide incorporation. This method provides a fast, accurate, and sensitive approach to sequencing, with applications in various fields of genetic and genomic research.

DNA Preparation:

• The DNA to be sequenced is fragmented, and adapters are attached to the ends of the fragments.

• A single-stranded DNA template is generated from these fragments.

1. Primer Binding:

   • A sequencing primer is hybridized to the single-stranded DNA template.

2. Enzyme Setup:

   • The sequencing reaction involves four key enzymes:

     • DNA Polymerase: Incorporates nucleotides into the growing DNA strand.

     • ATP Sulfurylase: Converts pyrophosphate (PPi) into ATP.

     • Luciferase: Uses ATP to produce light in the presence of luciferin.

     • Apyrase: Degrades unincorporated nucleotides and ATP, preparing the system for the next nucleotide addition.

3. Sequencing Reaction:

   • The DNA synthesis reaction is initiated by adding one of the four nucleotides (A, T, C, G) to the reaction mixture.

   • If the added nucleotide is complementary to the next base in the template strand, DNA polymerase incorporates it into the growing strand.

   • This incorporation releases pyrophosphate (PPi).

4. Detection:

   • The released PPi is converted to ATP by ATP sulfurylase.

   • The ATP then drives a reaction with luciferase, producing light.

   • The amount of light produced is proportional to the number of nucleotides incorporated.

   • This light signal is detected and recorded by a camera.

1. Signal Processing:

   • The intensity of the light signal is analyzed to determine how many nucleotides were incorporated.

   • If the added nucleotide is not complementary, no light signal is produced.

   • Apyrase then degrades the remaining nucleotides and ATP, resetting the system for the next nucleotide addition.

2. Iterative Process:

   • The process is repeated with each of the four nucleotides in a cyclical manner.

   • The sequence of the template strand is deduced from the pattern and intensity of the light signals generated during each nucleotide addition cycle.

Problem:

The following pyrogram has been created from a sequencing-by-synthesis procedure. Using your understanding of the procedure, identify the original sequence of the DNA fragment.

Solution:

The graph shows peaks where nucleotides were successfully incorporated into the growing DNA strand, with the height of each peak corresponding to the number of nucleotides incorporated. Therefore, the correct sequence would be A, T, G, G, G, G, G, A, A, T, G, T, T.

Author: Sanjay Adireddi

References: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727787/