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DNA sequencers automate the determination of the precise order of nucleotide bases—adenine, guanine, cytosine, and thymine—in a DNA region of interest. The order of the DNA bases is reported as a text string, called a read.
DNA sequencers have wide-ranging applications in molecular biology and genetics laboratories to further scientific progress or to aid in medical treatment decisions and genetic counseling. There are many DNA sequencers to choose from for small, medium, and high-throughput sequencing requirements.
DNA sequencing techniques
Sanger sequencing
The Sanger sequencing technique refers to capillary electrophoresis-based methods of DNA sequencing. The basic Sanger technique involves isolating the DNA sample and amplifying the DNA region of interest. The DNA sample is divided into four separate sequencing reactions, containing all four of the deoxynucleotides. Only one of the four corresponding DNA polymerases is added to each reaction to form an extension product with chain elongation occurring at the 3’ end of a primer.
Following amplification, sequencing products are generated by cycle sequencing, followed by purification of the reaction products to remove fluorescent ddNTP. The reaction products are separated by capillary electrophoresis and data are generated and analyzed.
Life Technologies Sanger sequencing kits use cycle sequencing protocols based on dye primer chemistry or dye terminator chemistry. In dye primer chemistry, fluorescently labeled fragments are generated by incorporation of the dye-labeled ddNTPs at the 5’ end of the fragment. In dye terminator chemistry, fluorescently labeled fragments are generated by incorporation of the dye-labeled ddNTPs at the 3’ end of the fragment.
BigDye® primers and terminators from Life Technologies use single energy transfer molecules. The energy transfer molecules include an energy donor and acceptor dye (a dRhodamine dye) connected by an energy transfer linker.
The 3130 and 3130xl Sanger sequencers utilize the BigDye Direct Cycle Sequencing Kit based on the dye primer chemistry, and the 310 Sanger sequencer uses the BigDye Terminator v1.1 Cycle Sequencing Kit based on the dye terminator chemistry.
The Sanger sequencing techniques have inherent limitations of scalability, throughput, speed, and resolution.
Next-generation sequencing
The concept of the next-generation sequencing (NGS) technique is the same as the Sanger technique, but extends the process of DNA sequencing across millions of reactions in a massively parallel fashion without being limited to a single or a few DNA fragments.
The NGS technique amplifies a single genomic DNA sample by first fragmenting it into a library of small segments, which are then sequenced into millions of parallel reactions. The strings of sequenced bases are reassembled with a known reference genome used as a scaffold (resequencing) or without a reference genome (de novo sequencing). The full set of aligned bases reveals the entire sequence of each chromosome in the genomic DNA sample.
Purchasing considerations for DNA sequencers
In choosing a DNA sequencer, many factors require consideration.
Application
Sanger sequencers support smaller-scale DNA sequencing, RNA sequencing, and epigenetic analysis with a minimal requirement of throughput and analysis time.
Life Technologies offers the 3730xL, 3730, 3500xL, 3500, 3130xL, 3130, and 310 Sanger sequencers with wide-ranging applications in cancer research, genetic disease research, agriculture biology research, food testing, and epigenetic analysis.
NGS systems support large-scale and automated genome analyses with the requirement for high throughput and analysis time.
Also offered by Life Technologies are the Ion Proton™ and Ion PGM™ NGS systems. The Ion PGM system provides analysis of retrospective samples for specific disease types, surveillance, investigating outbreaks, and determining disease etiology. The 5500xl SOLID™ sequencer for large genomic studies and 5500 SOLID sequencer for smaller-scale investigation rely on the SOLID NGS technique, which is a template-bead sequencing technique.
Illumina offers the MiSeqDx NGS system, which is FDA-cleared as an in vitro diagnostic and has numerous applications for targeted gene sequencing, metagenomics, small genome sequencing, targeted gene expression, amplicon sequencing, and HLA typing. The company also offers the MiSeq sequencer for de novo sequencing.
The PACBIO RS II from Pacific Biosciences is a Single Molecule, Real-Time (SMRT®) sequencer. SMRT technology records light pulses emitted as a by-product of nucleotide incorporation. It is suitable for de novo assembly, characterization of genetic variation, methylation analysis, and microbiology studies.
Siemens Medical Solutions USA, Inc. offers the OpenGene® DNA sequencing system for viral resistance and genotype sequencing. The TRUGENE® HIV-I genotyping kit runs on the OpenGene DNA sequencing system.
Throughput
NGS systems generally have a higher throughput than Sanger sequencers.
454 Life Sciences claims that the GS Junior Plus NGS system is capable of producing read lengths of 700–800 bases and longer, and the GS FLX System read lengths of up to 1 kb.
According to Life Technologies, the Ion PGM NGS system produces up to 6 million 400-base reads for 2 Gb max per hour run, and the Ion Proton NGS system produces up to 80 million 200-base reads for 10 Gb to 14 Gb per run. These systems rely on Ion AmpliSeq™ technology, which enables the amplification of thousands of targets using just 10 ng of DNA.
The MiSeq sequencer from Illumina enables up to 15 Gb of output with 25 M sequencing reads and 2 × 300 bp read lengths.
The SOLID systems from Life Technologies rely on massive parallelization and shorter read lengths to achieve ultrahigh-throughput sequencing. These systems can generate more than 1 Gb of useable data per run.
Sequencing time
NGS systems generally have a faster sequencing time than Sanger sequencers.
The HSeq 2000 from Illumina, GS FLX System, and 5500xl SOLID are NGS systems with the slowest sequencing time (about 8 days).
The GS Junior Plus, Ion PGM, Ion Proton, and MiSeq are NGS systems with the fastest sequencing time (<1 day).
Cost
The initial cost and the cost for running the sequencer are important considerations.
The HSeq 2000, GS FLX System, and 5500xl SOLID sequencers have the lowest cost per base. The GS Junior Plus, Ion PGM, and MiSeq have a minimum capital cost.
Customization
Most providers offer customization options.
The Ion Chef™ system from Life Technologies is intended to provide automation and high-throughput research for reproducible template preparation and chip loading for the Ion PGM and Ion Proton systems.
The PathAmp™ FluA reagents and Pathogen Analyzer Plugin for Torrent Suite™ Software paired with the Ion PGM system enable researchers to produce influenza A typing and analysis of retrospective influenza samples.
Representative DNA sequencers
- 454 Life Sciences: GS Junior Plus, GS FLX+ System
- Beckman Coulter, Inc.: GenomeLab™ GeXP
- Illumina: MiSeq, MiSeqDx, NextSeq 500, HiSeq 2500, HiSeq X Ten, NeoPrep
- Life Technologies: 3730xL, 3730, 3500xL, 3500, 3130xL, 3130, 310, Ion PGM, Ion Proton, 5500 SOLID, 5500xl SOLID
- Pacific Biosciences: PACBIO RS II
- Siemens Medical Solutions USA, Inc.: OpenGene
Table 1 – Providers of DNA sequencers
Developments to watch
Oxford Nanopore Technologies®, a developer of third-generation technologies based on nanopore electronic systems, offers the MinION® and GridION® systems for the analysis of DNA, RNA, and proteins. The devices can be used in scientific research, personalized medicine, crop science, security and defense, and environmental applications.
Providers
A list of providers of DNA sequencers is given in Table 1.
Lina Genovesi, Ph.D., JD, is a Technical, Regulatory, and Business Writer based in Princeton, NJ; e-mail: [email protected]; www.linagenovesi.com
Please see our Automated DNA Sequencer / DNA Sequencing System to find manufacturers that sell these products