MiSeq System

TM

Sole Source Specifications

The Illumina MiSeq personal sequencing system enables researchers to go from sample to analyzed data in as little as eight hours with a revolutionary workflow, unmatched accuracy, and flexibility to undertake the widest range of applications. Capable of generating greater than 6 Gb per run, MiSeq is the only next-generation sequencer that integrates amplification, sequencing, and data analysis in a single instrument with a footprint of less than 2 feet square. MiSeq leverages Illuminas proven TruSeq chemistry for workflow simplicity and unmatched accuracy, making it the ideal platform for rapid, cost-effective genetic analysis.

Table of Contents
System Workflow and Applications Sequencing by Synthesis Sample Preparation Amplification Applications 1 3 5 6 7

System Workflow and Applications Feature

Fastest turnaround time from sample to data

Description
< 8 hour turnaround time from purified DNA to direct reporting of variants 2-hour library prep(15minutes hands on ) 1-hour on-instrument clonal amplification 3-hour on-instrument sequencing run (1 36 bp) 2-hour on-instrument analysis for variant reporting

Integrated workflow

Single, integrated instrument Single instrument performs clonal amplification, sequencing, and data analysis (base calling, alignment, variant calling) Complete amplification, sequencing and data analysis in less than 2 feet square instrument space No need for dedicated ancillary amplification system or computing infrastructure Does not require emulsion PCR

Lowest hands-on time

Walk-away operation with as little as 50 minutes total hands on time from sample to analyzed data As low as 1530 minutes total hands-on time for library prep 20 minutes hands-on time for run set up (amplification, sequencing, paired-end) Intuitive touch screen user interface, RFID-tracking, premixed/pre-loaded integrated reagent cartridge Paired-end capable; integrated paired-end fluidics on instrument Supports up to 2 150 bp read length, upgradable to 2 x 250bp Greater than 5Gb of high-quality data passing filter per 2 150 bp /2 x 250bp run

PE capability

Highest output

Highest data quality

Accurate variant detection enabled by proven SBS chemistry Uses Illuminas reversible-terminator sequencing by synthesis (SBS) chemistry >85% of bases with Q scores greater than 30 (1 35 bp) Competitive addition from a pool of all four reversible terminator nucleotides labeled with four different fluorescent dyes eliminates homopolymer errors

Widest application flexibility

Flexible and Simple integrated solution for targeted resequencing Supports TruSeq custom targeted enrichment kits and assays A range of optimized targeted resequencing assays interrogate 10s to 100s of targets per sample quickly and easily Variable length amplicon sequencing spanning 100s to 1000s of base pairs Highly multiplexed PCR amplicon sequencing plate-based processing of 96 samples per plate and up to 384 amplicons per sample

Widest application flexibility among all personal sequencers Greater than 6 million reads per single-end run enable CHiP-Seq and small RNA applications Greater than 6 Gb of output allows multiplexed sequencing of viral and bacterial genomes in a single run Greater than 1,000 peer-reviewed publications using Illuminas reversible terminator-based sequencing by synthesis chemistry

Sequencing by Synthesis Description

Data Generation

Features
Number of reads Greater than 7 million clusters passing filter enable the system to generate greater than 7 million reads per single-end run, or greater than 14 million reads per paired-end run in a single flow cell

Throughput for a single flow cell Greater than 6 Gb of high-quality passing filter data per 2 250 bp run ,

Sequencing Flexibility

Sequential interrogation of bases allows flexible adjustment of read length during a run Supports read lengths up to 2 250 bp run

Instrumentation

MiSeq physical specifications Benchtop instrument; W D H: 68.6 cm 56.5 cm 52.3 cm (27.0 in 22.2 in 20.6 in)

Illumination Light-emitting diode at 530 nm, 660 nm

Reagent handling Reagent chiller compartment has capacity for one integrated reagent cartridge containing reagents for cluster generation, paired-end chemistry, and at least 300 cycles of sequencing Reagents come premixed and preloaded in an integrated reagent cartridge

Flow cell loading Flow cells are auto-positioned and held in place by a clamping mechanism

Instrument control computer On-instrument data analysis including base calling, alignment, and variant calling Variant reporting occurs within 2 hours of run completion

Flow cells Chemistry MiSeq is a single flow cell system Each flow cell is a substrate with a single channel

Illuminas sequencing by synthesis chemistry uses reversible terminators and a highly efficient DNA polymerase modified to accept reversible terminator nucleotides. Sequencing reactions on up to 12 samples per flow lane is performed in a self-contained single channel flow cell environment The DNA polymerase is modified for efficient addition of nucleotides with cleavable fluorescent dyes and reversible terminators Fluorescent dyes on the nucleotides are cleaved after imaging The reversible terminators are removed to allow chain extension Competitive addition from a pool of all four reversible terminator nucleotides labeled with four different fluorescent dyes eliminates homopolymer errors Sequenced DNA templates are be copied to generate complementary strand, enabling paired-end sequencing Forward DNA strands are be selectively washed out of the flow cell

Sequencing Run Time & output

Sequencing runs* can be completed in:

Cluster Generation and Sequencing

READ LENGTH (BP)

TOTAL TIME*

OUTPUT

CURRENT

FUTURE**

CURRENT

FUTURE**

1 36 2 25 2 100 2 150 2 250

4 hrs 5 hrs 19 hrs 27 hrs NA

3.2-3.5 hrs 4.6-5 hrs 14-16 hrs 20.7-24 hrs >35 hrs

175-245 Mb 250-350 Mb 1.0-1.4 Gb 1.5-2.0 Gb NA

440-550 Mb 640-800 Mb 2.5-3.1 Gb 3.7-4.6 Gb 6.0-7.0 Gb

*Includes paired-end sequencing read, if applicable. **Performance, output, amplification, and sequencing time indicated as "Future" applies to the next version of the instrument that is anticipated to be commercially available in the third quarter of 2012. Customers will be notified of availability of this next version of the instrument, dates are subject to change.

On-Instrument Analysis

READ LENGTH

APPROXIMATE DURATION

Paired-End 150 Cycles (2 150 bp)

Less than 2 hours for all supported analysis workflows, except 16s metagenomics. 16s metagenomics completes in less than 24 hours.

Reads Passing Filter

RUN TYPE

READS PASSING FILTER (MILLION)*

CURRENT

FUTURE**

Single Reads Paired-End Reads

5-7 M 10-14 M

12-15 M 24-30 M

*Includes paired-end sequencing read, if applicable. **Performance, output, amplification, and sequencing time indicated as "Future" applies to the next version of the instrument that is anticipated to be commercially available in the third quarter of 2012. Customers will be notified of availability of this next version of the instrument, dates are subject to change.

Quality Scores*

READ LENGTH

BASES HIGHER THAN Q30

1 36 bp 2 25 bp 2 100 bp 2 150 bp
Q-SCORE

> 90% > 90% > 85% > 75%

CHANCE OF WRONG BASE CALL

Q40 Q30 Q20 Q10

1 error in 10,000 1 error in 1,000 1 error in 100 1 error in 10

A quality score (Q-score) is a prediction of the probability of an error in base calling.

Instrument Control Software

MiSeq Control Software (MCS) offers a simple interface to configure, launch, and monitor runs. Standardized data format allows for the seamless use of downstream analysis and visualization tools.

Sample Preparation Description

Sample Preparation Requirements Sample Preparation Kits

Features
Genomic DNA sample libraries can be prepared in 2 hours or less by one full-time employee (FTE) for both single and short-insert (<500 bp) paired-end runs.

Ready-to-use kits are available to prepare samples for: DNA sequencing (single- or paired-end reads and mate pairs) Transcriptome sequencing (RNA-Seq) Validated disease pannel Sample multiplexing o 12 indexes per lane currently supported o Highly multiplexed amplicon sequencingup to 384 targets per sample, 96 samples per run

Plus additional applications as developed

Paired-End Read Support

Kits enable a unique combination of paired-end insert size ranges:

200500 bp (short insert paired-end) 210 kb (long insert mate pairs) < 1 g DNA for genomic DNA sequencing applications (as low as 50 ng for many genomic DNA samples) As low as 10 ng DNA for ChIP-Seq applications < 1 g total RNA for small RNA sequencing applications < 1 g Total RNA for mRNA-Seq applications (as low as 100 ng for many total RNA samples) < 1 g DNA for bisulfite-converted DNA sequencing applications

Low Sample Input

Amplification Description
Amplification Method

Features
Solid-phase isothermal amplification to produce clonal single-molecule array clusters is completely automated, requiring no user intervention. No need for emulsion PCR or additional pieces of equipment amplification is performed on-same instrument.

Amplification Sample Throughput Amplification Time

Genomic DNA sample library can be prepared in 2 hours or less by one full-time employee (FTE) for both single and short-insert (<500 bp) paired-end runs.

A single operator can amplify up to 12 samples or more on a single channel flow cell in one hour using a single MiSeq instrument. Amplification is fully automated, using an integrated reagent cartridge with premixed and loaded reagents. MiSeq performs automated simultaneous clonal amplification of hundreds of millions of single molecule DNA templates producing clusters containing approximately 1,000 identical copies of each original DNA sequence.

Cluster Generation

Applications Description
Customerdemonstrated applications using Illuminas reversible terminator-based sequencing by synthesis chemistry with peer-reviewed publications.

Features
Whole-genome resequencing Targeted resequencing including, but not limited to the following methods: o TruSeq custom enrichment o TruSeq custom amplicon sequencing o Long-range PCR o Molecular inversion probes De novo sequencing ChIP-Seq of sequence-specific DNA binding proteins ChIP-Seq of histone modifications and epigenetic marks Sequencing of bisulfite-treated DNA to study DNA methylation Metagenomics (16S rRNA-sequencing) mRNA sequencing Tag-based gene expression Small RNA sequencing Ribosome profiling DNAse 1 hypersensitivity site mapping Nucleosome positioning and chromatin structure studies CLiP-Seq: studying sequence specific protein-RNA interactions CNV-Seq: measuring copy number variation with sequencing GRO-Seq: studying RNA polymerase initiation events Sequencing of ancient DNA samples Paired-end mRNA sequencing DNA imprinting and allele specific expression Plus additional applications as developed.