Exploring the microbial metagenome for food security using MiSeq next-generation high-throughput DNA sequencing

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The Illumina MiSeq system is a "sequencing-by-synthesis"-based, next-generation high-throughput DNA sequencing platform. A single instrument performs clonal amplification, sequencing, and data analysis (base-calling, alignment, variant calling, and reporting). DNA or cDNA fragments of interest are ligated to specific oligos called adapters, which enable hybridization of the template to the surface on which the sequencing reaction occurs (single microfluidic cell). The adapters also contain PCR and sequencing primer annealing sites and 6-base indexing that allows usage of more than one sample per run. The DNA strands are amplified by bridge amplification, forming millions of discrete clusters of clonal copies of DNA. During the sequencing run, each cluster becomes visible as a small white speck due to the fluorescence that is emitted by fluorescently labeled dd-NTPs which are slightly-modified bases that prevent the polymerase from adding another nucleotide to the growing chain after the first one has been added (termination of extension). A laser excites the green fluorophore and a CCD camera captures the emitted fluorescence. This is followed by cleavage of the fluorophore and removal of the terminator, so that another base can be add to the growing chain. The MiSeq is a massively-scalable platform that provides 2x300 bp reads of the highest quality data and up to 50 million paired-end reads, generating up to 15 Gb of data. Very reliable homopolymer length measurements can be obtained with MiSeq, since the Illumina system only incorporates one nucleotide at a time and has a reversible terminator step. The Illumina MiSeq platform will be used by the applicants in support of their applied microbiological, innovative food security bioproducts, basic and advance ecological research to perform: i) genome sequencing, ii) metagenomic applications, such as using targeted phylogenetic primers for functional and biodiversity amplicon analysis of complex microbial communities, and iii) transcript gene expression as well as whole transcriptome analyses. There is presently no comparable high-throughput sequencer available on the market.