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

Illumina MiSeq系统是一个基于“合成测序”的下一代高通量DNA测序平台。单个仪器执行克隆扩增、测序和数据分析(碱基调用、比对、变体调用和报告)。将感兴趣的DNA或cDNA片段连接到称为接头的特定寡核苷酸上，这使得模板能够与发生测序反应的表面(单个微流控细胞)杂交。该适配器还包含聚合酶链式反应和测序引物退火点和6碱基索引，允许每次运行使用一个以上的样本。DNA链通过桥式扩增进行放大，形成数百万个离散的DNA克隆拷贝簇。在测序过程中，由于荧光标记的dd-NTP发出的荧光，每个簇都变得可见为一个小的白色斑点，这些dd-NTP是略微修饰的碱基，防止聚合酶在第一个核苷酸添加到增长的链中后添加另一个核苷酸(延伸终止)。激光激发绿色荧光团，ccd摄像机捕捉发射的荧光。紧随其后的是切割荧光团和去除终止子，这样就可以在不断增长的链上添加另一个碱基。MiSeq是一个可大规模扩展的平台，可提供2x300个BP的最高质量数据读取和多达5000万个成对端读取，从而生成多达15 GB的数据。使用MiSeq可以获得非常可靠的均聚物长度测量，因为Illumina系统一次只包含一个核苷酸，并且具有可逆的终止子步骤。Illumina MiSeq平台将被申请者用于支持他们的应用微生物学、创新的食品安全生物技术产品、基础和高级生态学研究，以执行：i)基因组测序，ii)元基因组应用，例如使用定向的系统发育引物进行复杂微生物群落的功能和生物多样性扩增分析，以及iii)转录基因表达以及整个转录组分析。目前市场上还没有类似的高通量测序仪。

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.