Chapter 6 : Next-Generation Sequencing

Sequencing methods for currently available NGS platforms. (A) The sequencers manufactured by Roche/454 (left), SOLiD (middle), and Ion Torrent (right) all use bead-based emulsion PCR (rectangular inset) in the library generation process, followed by different approaches to fluorescent-based sequencing. (B) Illumina sequencing involves library generation on a flow cell via a sequencing-by-synthesis approach and the imaging of millions of fluorescent flow cell clusters. (C) PacBio sequencing is performed by a DNA polymerase enzyme affixed to a glass substrate in a zero-mode waveguide nanostructure. Each nanostructure generates an individual sequence. (D) Nanopore sequencing, as performed by the Oxford Nanopore MinION instrument, leverages the voltage conductance changes (left) that occur in response to passage of DNA through a nanopore (right), a protein in the lipid-bilayer membrane containing a single hole that allows a single molecule of DNA to pass through.

Schematic overview of an NGS pipeline. Sample processing for NGS involves a stepwise process of nucleic acid extraction, library preparation, and sequencing on a dedicated instrument (left). Following generation of raw data, bioinformatics analysis of metagenomic or microbial NGS data includes preprocessing, de novo assembly, host subtraction, pathogen identification, taxonomic classification, and results reporting/visualization (right). The asterisks denote optional steps in the procedure.