DNA >Sequencing
MAX-Seq™ Genome Sequencer
#Sequencing by synthesis
#High throughput : 2 flow cells with 8 lanes
#Key applications:
- ChIP-seq
- mRNA-seq
- small RNA-seq
- DNA re-sequencing
- de novo sequencing (RNA or DNA)
- targeted DNA-seq
- Whole-Exon-seq
#Read Length
- 35 pb
- 55 pb
- 100 pb currently under development
MAX-Seq™ is based on the shot-gun sequencing technology, developed in the late 90s by the Church group. With “shot-gun” technology, a whole genome is fragmented into billions of short overlapping fragments and amplified. These small overlapping fragments make up the “library” for the particular genome. The genome library is sequenced and then the sequences for all of the short fragments are aligned by a computer against a reference genome, so that any differences between the sample and the reference may be identified and changes (mutations, SNPs, re-arrangements, etc.) discovered.
To perform 2nd generation sequencing with the
MAX-Seq™ Sequencer using shot-gun
sequencing technology, the following steps are
used:
• Create a library for the genomic sample
• Sequence the library on the MAX-Seq™
• Analyze the data by either mapping to a
reference genome or de-novo assembly
Library Construction
The first step in sequencing a genome using the
MAX-Seq™ sequencer is to break the genome
into billions of small overlapping fragments.
The MAX-Seq™ supports both bead-based
library construction and DNA nanoball library
construction protocols. Both protocols require
the fragmenting of your genome into small
fragments and attaching of adapters (primer
sites) to the end of each of the DNA fragments.
With bead-based library construction, each fragment of the library is individually amplified onto a bead using emulsion PCR; whereas, with
DNA nanoball library construction, the individual library fragments are circularized and then amplified using rolling circle amplification
to generate compact submicron spheres of DNA for each library fragment.
Sequencing the Library using Sequencing by Synthesis (SBS)
Once a library has been created, the next step in
the analysis is the sequencing step. This is where
the power of the MAX-Seq™ using Sequencing
by Synthesis takes over.
Sequencing by synthesis is a cyclic process
involving the use of polymerase that
incorporates 3’ reversibly blocked, dye labeled
deoxynucleotide triphosphates (dNTPs). Each of
the four nucleotides, A, C, T and G, have a
unique fluorescent dye color. The dyes allow the
query of the base incorporated by polymerase.
The steps for sequencing by synthesis are:
1. Hybridize the sequencing primer
2. Add a mixture of four 3’ reversibly blocked, dye-labeled dNTPs, polymerase and buffer
3. Let the polymerase incorporate the complimentary base into the chain (EXTEND)
4. Wash the excess polymerase and unincorporated dNTPs away.
5. Read the dye color on the dNTP that was incorporated into each fragment. (MEASURE)
6. Cleave off the dye.
7. Cleave off the blocking group. (CLEAVE)
8. Repeat the process for the desired number of cycles, moving down the DNA template one base at a time.
Process
All the library construction steps are done in tubes. The flow cell are used only in sequencing part.
Workflow :
1. Install one or two flow cells - After preparing the library and loading it into a flow cell, simply slide the flow cell into the carrier.
2. Load the SBS reagents - The SBS reagents are provided in several 50 ml-format tubes and washing buffers in 1 liter bottles.
3. Program the software for the number of flow cells, lanes and number of cycles.
4. Press Start.
After the start button is pressed, the MAXSeq ™ sequencer then does everything else.
The MAX-Seq™ Instrument
The MAX-Seq™ is a very sensitive, highly automated instrument for performing automated sequencing and analysis of DNA.
Optics
• 5 Mp CCD electronic camera
• Autofocus Xenon excitation lamp
• 6 position filter wheel (4 used)
• High Quality Fluorescent Microscope Optical Bench
• Frictionless, Magnetic Highly Accurate Stage Movement
Fluidics
• SBS optimized fluidics
• Syringe Pump Delivery
• Dual 8 lane flow-cell design
• Compartments for Cooled and room temperature reagents
Instrument dimensions: 84 cm x 76cm x 76cm.
Weight : 160kg
