Next Generation Sequencing

Next Generation Sequencing

Next Generation Sequencing is an advanced sequencing technology where many short DNA molecules are sequenced at the same time. The technology is also called massively parallel sequencing. These short DNA molecules are then assembled by comparing their sequence to a reference sequence, thereby revealing the complete DNA sequence that can be very long (as long as our genome!).

There are many different platforms of Next Generation Sequencing, and each of them utilizes a different technique in achieving DNA sequencing. In this case, we will focus on the reversible dye termination technology employed by Illumina. In this method, the DNA is first fragmented into smaller sizes, and two short DNA molecules called "Adapters" are ligated to each end of the sample (see Figure 1). These adapters will function as primer-docking sites to amplify the DNA during PCR and to bind to the flow cell. Before analyzing the data, we remove the adapters because they are not biological sequences.

DNA molecules capped with adapters and primers are first attached to a slide (called the flow cell) and amplified with the polymerase enzyme creating local clonal DNA colonies. These DNA colonies are also referred to as DNA clusters. Each of the clusters contains the same DNA sequence, hence the term clonal DNA colonies. Similar to the First Generation Sequencing technique, the nucleotides are individually labeled with a fluorescent dye. After the addition of a nucleotide, the elongation stops, and a picture is taken. Following this, the blocker sitting at the 3' terminal is then chemically removed from the DNA, allowing the next cycle of nucleotide addition to start.

Once the DNA is extracted, it needs to be processed in preparation for sequencing. The different sample preparation steps are outlined below:

These steps are followed by Cluster generation and the actual Sequencing process.

Visual representation of the next generation sequencing workflow. First, DNA, which is represented by two parallel lines that form a curving, tubular shape, is sheared to form many short, straight fragments of parallel lines. The next step is to select approximately 200 to 300 base pair fragments. Next, adapters are attached the fragments to form a sequencing library. This is represented by red DNA sections being attached to both ends of the base pair fragments. Then, the sequencing library is applied to a flowcell, which is represented by a green rectangle containing several horizontal rows that run the length of the shape. Next, cluster generation by solid PCR, or bridge amplification, shows DNA being replicated by base pairs aligning along a curved DNA fragment to form base pairs. The sample is then analyzed by the next generation sequencer. NGS performs sequencing by synthesis with reversible terminators.

Figure 1. Workflow of Next Generation Sequencing using Illumina platform.

The parallel sequencing produces billions of reads per run. This is exponentially larger than the reads that are provided by First Generation Sequencing. Next Generation Sequencing is a robust technique that can be used for many different applications, such as SNP profiling, gene expression analysis, detecting genetic aberrations such as mutation or chromosomal rearrangements.

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