​Illumina Sequencing

 

The MiSeq sequencer is capable of automated paired-end reads and up to 15 Gb per run, delivering over 600 bases of sequence data per read. The library prep kits that it uses are optimized for a variety of applications, including targeted gene, small genome, and amplicon sequencing, 16S metagenomics, and more.

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Principle of operation

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Sequencing by Synthesis

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A fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.

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The end result is true base-by-base sequencing that enables accurate data for a broad range of applications. The method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers).

 

Key applications and methods

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Small Genome Sequencing

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Get a detailed genome view of the smallest organisms. Small genome sequencing provides comprehensive analysis of microbial or viral genomes for public health, epidemiology, and disease studies. Sequence up to 24 small genomes per MiSeq run.

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• Nextera XT Library kit prepares sequencing libraries for small genomes, PCR amplicons, and plasmids in less than 90 min, with a low DNA input requirement

• MiSeq Reagent Kit v3 (600 cycles)

• SPAdes Genome Assembler- open source tool for de novo sequencing, designed to assemble small genomes from MDA single-cell and standard bacterial data sets

 

Targeted Gene Sequencing

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Targeted resequencing focuses time, expenses, and analysis on sequencing only a subset of genes or genome regions of research interest. Amplicon sequencing, the ultra-deep sequencing of PCR amplicons, enables cost-effective analysis of up to hundreds of target genomic regions in one assay. Sequence up to 96 samples in a single MiSeq run.

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• AmpliSeq for Illumina Library kit uses a highly multiplexed PCR-based workflow for use with targets ranging from a few to hundreds of genes in a single run

• MiSeq Reagent Kit v2 (300 cycles). Micro and nano formats are available for low output applications

• BaseSpace Variant Interpreter enables researchers to rapidly identify biologically significant variants from human genomic data

 

16S Metagenomic Sequencing

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Sequencing the 16S ribosomal RNA (rRNA) gene is a culture-free method to identify and compare bacteria from complex microbiomes or environments that are difficult to study. Our demonstrated protocol for 16S rRNA sequencing can help take the guess work out of your experiments. Multiplexing lets you sequence up to 96 samples per MiSeq run.

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• Nextera XT Index Kit (v2) allow for up to 384 uniquely indexed samples to be pooled and sequenced on a single sequencing run

• MiSeq Reagent Kit v3 (600 cycles)

• 16S Metagenomics BaseSpace App performs taxonomic classification of 16S rRNA targeted amplicon reads using an Illumina-curated version of the GreenGenes taxonomic database