Illumina MiSeq Next Generation Sequencer

The MiSeq is an integrated instrument that performs clonal amplification, genomic DNA sequencing, and data analysis with base calling, alignment, variant calling, and reporting in a single run.  The MiSeq benchtop instrument utilizes a double-sided, single-lane flow cell and reagent cartridge supplied in kit form.  Sequencing is performed by recording the synthesis of DNA strands in clusters of sample templates attached to the flow cell.  Each newly attached base liberates a fluorescent dye that is excited by diode lasers (530 & 660 nm) and imaged using two digital cameras.  Sequential interrogation of bases allows for the flexible adjustment of read length during a run.  Up to 96 samples may be sequenced in a single run with DNA libraries prepared with indexed or bar-coded adapters.  The capacities of the single lane flow cell and the possible MiSeq applications are shown in the following tables.

Read Length (bp)

Time (hrs)

Data Output

1 x 35

~4

600 Mb

2 x 25

~5

800 Mb

2 x 75

~24

3.7 Gb

2 x 150

~24

5.0 Gb

2 x 250

2 x 300

~39

~65

8.0 Gb

15 Gb

 

MiSeq Workflow

Applications

File Format

Assembly

Assembles small genomes from reads without the use of a genomic reference. contigs.fa

Custom amplicon (human only)

Sequences TruSeq Custom Amplicon from probes targeting particular genome positions and aligns reads against a specified manifest file. *.bam; *.vcf

FASTQ only

Generates intermediate analysis files in FASTQ format enabling the use of third-party tools to analyze sequencing data. *fastq.gz

Library QC

Aligns reads against specified reference genomes and generates a sample report. *.bam

Metagenomics

Classifies bacteria from a metagenomic sample of amplified 16S rDNA. *.txt.gz

PCR amplicon

Sequences PCR amplicons (generally prepared using Nextera XT tagmentation). Aligns reads against specified reference genomes and performs variant analysis for the regions specified in the manifest file. *.bam; *.vcf

Resequencing

Sequences a small genome, aligns reads against a specified reference genome, and performs variant analysis. *.bam; *.vcf

Small RNA

Sequences miRNA and aligns reads against databases for contaminants, mature miRNA, small RNA, and a genomic reference. *.contam; *.genome; *.mirna; *.rna