About Next-Generation Sequencing

The first-generation Sanger sequencing method has inherent technical limitations including low-throughput data generation, slow speed, high cost, and difficulty to analyze allele frequency. To achieve the goal of a $1,000 human genome, new sequencing technologies have been coming out since 2004. These technologies are massively parallel, thereby achieving high-throughput data generation. The streamlined sample preparation step prior to sequencing leads to significant savings in time and cost. Since each sequencing reaction is carried out on one fragment of DNA, different alleles can be analyzed at the same time.

Sequencing Workflow

Process of library preparation and sequencing. Adaptors are added to DNA fragments, which bind to a flow cell. The other end of the fragment bends to also bind to the flow cell, where the fragment is then extended. Doing this many times forms clusters. Sequencing identifies each nucleotide based on a different fluorescent color, and images the flow cell after each nucleotide.
Yuan Lu, Yingjia Shen, Wesley Warren and Ronald Walter (January 14th 2016). Next Generation Sequencing in Aquatic Models, Next Generation Sequencing - Advances, Applications and Challenges, Jerzy K Kulski, IntechOpen, DOI: 10.5772/61657.

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Library Preparation

Adds adapters and indexes ("barcodes") to DNA fragments so they can be pooled with other samples and identified after sequencing

 

Different Strategies:

 

Bead-linked transposome tagmentation:

DNA is fragmented and normalized by binding to bead transposomes

 

Adapter ligation:

genomic DNA or cDNA is fragmented and has adapters attached to both ends of the fragments

 

Amplicon prep:

amplify DNA/cDNA, remove primer sequences, ligate sequencing adapters and amplify

Cluster Generation

Process by which libraries bound to the surface of the flow cell are clonally amplified (bridge amplification)

Unbound end of fragment binds to the flow cell to form a "bridge". This bridge is then amplified to generate an identical fragment which can also undergo bridge amplification

Sequencing

Fluorescently labeled nucleotides are incorporated and imaged one base at a time as they are added to the clusters of fragments on the flow cell. The sequencer uses the wavelength/intensity of light emission from each cluster to identify the nucleotide.