The Power of Next-Generation Sequencing in Your Hands
On the Path towards Diagnostics
• Proven technology: Uses the same long read GS FLX Titanium chemistry as the GS FLX System to deliver high-quality, bioinformatics-friendly long reads.
• Accessible : Small instrument size, along with low entry and operating costs, enables any lab to take advantage of next-generation sequencing.
• Easy to use : Move quickly from data to results using the comprehensive suite of dedicated data analysis software.
GS Junior Titanium Reagents & Accessories
Benchtop Instrument
& Computer
Data Processing
& Analysis Software
An Integrated Solution – from Sample Prep to Data Analysis GS Junior System Long Reads
GS Junior System
System Type Benchtop instrument
Throughput ~35 Mb
HQ Reads per Run ~70,000 amplicon, 100,000 shotgun Average Read Length ~400 bases
Accuracy Q20 read length of 400 bases (99% accuracy at 400 bases and higher for preceding bases) Run Time 10 hours
Sample Input Genomic DNA, PCR products (amplicons) or cDNA, depending on the application
Computing High-performance desktop PC for run processing and analysis included
Data Analysis Software
GS Reference Mapper GS De Novo Assembler GS Amplicon Variant Analyzer Dimensions 40 cm x 60 cm x 40 cm
Weight 25 kg
Power Supply 100 VAC-240 VAC 50/60 Hz 400 VA
Typical performance. Actual results depend on specifi c sample and genomic characteristics.
Example read length distribution from a GS Junior instrument run.
Average read length is 488 bp with a modal read length of 496 bp.
The GS Junior System
The Power of Next-Generation
Sequencing on Your Benchtop
From Sample to Result in 72 Hours
The Complete Workfl ow
Isolate DNA from any kind of sample, such as blood or tissue, using manual or standardized automated protocols yielding high quality nucleic acids. Prepare your amplicon or shotgun sequencing library yet the same day and clonally amplify the library overnight using the emPCR procedure. Start sequencing your samples on day two and analyze the results in depth with powerful and user-friendly bioinformatics software tools developed by 454 on day three. See page 4 for a detailed description of the 454 Sequencing workfl ow.
Sample Diversity
Highest Nucleic Acid Quality using Standardized Protocols
Sequencing on the GS Junior System
Digital Data Output of the Individual Long Reads
Manual sample prep (Roche High Pure) or
MagNA Pure Compact (8 samples)
MagNA Pure LC 2.0 (32 samples)
MagNA Pure 96 (96 samples)
Day 1
Day 2
Day 3
1. Library Preparation
• For shotgun librairies, start with as little as 500 ng of sample DNA, fragment using nebulization, and ligate Rapid Library Adapters to enable subsequent purifi cation, quantitation, amplifi cation and sequencing steps.
• For amplicon libraries, create PCR products by amplifying with specifi c fusion primers containing 454 Sequencing adaptor sequences.
2. emPCR Amplifi cation
• Anneal the library to 454 DNA Capture Beads and create water-in-oil emulsion to trap individual beads in amplifi cation microreactors.
• Amplify the entire emulsion in parallel to create millions of clonal copies of each library fragment on each bead.
3. Sequencing
• Load the beads onto the PicoTiterDevice – which allows only one bead per well – and place it into the GS Junior Instrument along with sequencing reagents.
• Start the sequencing run – individual nucleotides are fl owed in sequence across the wells. Each incorporation of a nucleotide complementary to the template strand on any given bead results in a chemiluminescent light signal recorded by the camera.
4. Data Processing & Analysis
• Process the raw data automatically on the accompanying desktop computer – the software uses the signal intensity at each well position to determine the sequence of 70,000 amplicon, or 100,000 shotgun reads simultaneously.
• Analyze results in depth with powerful and user-friendly bioinformatics software, e.g. for amplicon variant analysis.
454 Sequencing Workfl ow
Four Steps to Meaningful Results
TTCMRCARAYTTRG 1: ···AA··A·C··A·
2: ···CA··A·C··A·
3: ···AG··A·C··A·
4: ···CG··A·C··A·
···
31: ···AG··G·T··G·
32: ···CG··G·T··G·
Physically separate alleles by cloning (insert, grow, pick, grow, isolate plasmid
DNA, sequence)
Digitally separate alleles with AVA software (click)
4+ days
seconds
Capillary Sequencing
454 Sequencing Systems
Targeted Resequencing
Detect Variants at High Resolution
Zoom into critical genomic regions using amplicon sequencing of PCR products and sequence capture technologies. Quickly perform haplotyping, genotyping, rare variant detection, struc- tural variant detection, and heterozygote calling. Analyze disease-associated regions in oncology and immunogenetics, or viral quasispecies present within infected populations in infectiology.
The Long Read Advantage
• Long reads enable long amplicons – Link distant variants in regions up to 400 bp long with a single amplicon for high-resolution haplotyping, or even larger regions with tiled amplicons.
• Clonal reads without cloning – Long, clonal reads provide results in just a few days by parallel-processing thousands of amplicons. Detect vari- ants to < 1% frequency without the need to individually clone and sequence each sample, a process that takes weeks with standard technologies.
Applications
• Unambiguously resolve highly complex genomic regions (e.g., HLA, IgH) (Figure 1)
• Discover germline or somatic mutations in – oncology (e.g., EGFR, KRAS, BRAF, PI3K, BRCA) – hematology (e.g., TET2, CBL, RUNX1, RAS) – metabolic diseases (e.g., CFTR, MODY)
• Detect low-frequency variants such as rare drug- resistant viral mutations (e.g., HIV)
Figure 1. Non-clonal sequencing of amplicons can lead to ambiguous results due to the diffi culty in confi dently haplotyping loci with more than one variant. For example, this short 14-base region of the HLA DQA1 exon 2 locus contains fi ve variants resulting in 32 possible allelic combinations. Haplotyping involves physical separation of the two alleles by cloning, a time-consuming and labor-intensive process.
In contrast, the 454 Sequencing approach yields hundreds to thousands of clonal reads for each amplicon. Haplotyping can be accomplished completely in software without any need for additional sequencing. In addition, the large number of clonal reads also allows for confi dent calling of low-frequency variations.
Unambiguous Haplotyping
A key
MID
B MIDkey