DNA sequencer
A DNA sequencer is a
The first automated DNA sequencer, invented by
The
More recent, third-generation DNA sequencers such as PacBio
Because of limitations in DNA sequencer technology, the reads of many of these technologies are short, compared to the length of a
History
The first
More recently, a third generation of DNA sequencers was introduced. The sequencing methods applied by these sequencers do not require DNA amplification (polymerase chain reaction – PCR), which speeds up the sample preparation before sequencing and reduces errors. In addition, sequencing data is collected from the reactions caused by the addition of nucleotides in the complementary strand in real time. Two companies introduced different approaches in their third-generation sequencers. Pacific Biosciences sequencers utilize a method called Single-molecule real-time (SMRT), where sequencing data is produced by light (captured by a camera) emitted when a nucleotide is added to the complementary strand by enzymes containing fluorescent dyes. Oxford Nanopore Technologies is another company developing third-generation sequencers using electronic systems based on nanopore sensing technologies.
Manufacturers of DNA sequencers
DNA sequencers have been developed, manufactured, and sold by the following companies, among others.
Roche
The 454 DNA sequencer was the first next-generation sequencer to become commercially successful.[10] It was developed by 454 Life Sciences and purchased by Roche in 2007. 454 utilizes the detection of pyrophosphate released by the DNA polymerase reaction when adding a nucleotide to the template strain.
Roche currently manufactures two systems based on their pyrosequencing technology: the GS FLX+ and the GS Junior System.[11] The GS FLX+ System promises read lengths of approximately 1000 base pairs while the GS Junior System promises 400 base pair reads.[12][13] A predecessor to GS FLX+, the 454 GS FLX Titanium system was released in 2008, achieving an output of 0.7G of data per run, with 99.9% accuracy after quality filter, and a read length of up to 700bp. In 2009, Roche launched the GS Junior, a bench top version of the 454 sequencer with read length up to 400bp, and simplified library preparation and data processing.
One of the advantages of 454 systems is their running speed. Manpower can be reduced with automation of library preparation and semi-automation of emulsion PCR. A disadvantage of the 454 system is that it is prone to errors when estimating the number of bases in a long string of identical nucleotides. This is referred to as a homopolymer error and occurs when there are 6 or more identical bases in row.[14] Another disadvantage is that the price of reagents is relatively more expensive compared with other next-generation sequencers.
In 2013 Roche announced that they would be shutting down development of 454 technology and phasing out 454 machines completely in 2016 when its technology became noncompetitive.[15][16]
Roche produces a number of software tools which are optimised for the analysis of 454 sequencing data.[17] Such as,
- GS Run Processor converts raw images generated by a sequencing run into intensity values.[18] The process consists of two main steps: image processing and signal processing. The software also applies normalization, signal correction, base-calling and quality scores for individual reads. The software outputs data in Standard Flowgram Format (or SFF) files to be used in data analysis applications (GS De Novo Assembler, GS Reference Mapper or GS Amplicon Variant Analyzer).
- GS De Novo Assembler is a tool for de novo assembly of whole-genomes up to 3GB in size from shotgun reads alone or combined with paired end data generated by 454 sequencers. It also supports de novo assembly of transcripts (including analysis), and also isoform variant detection.[17]
- GS Reference Mapper maps short reads to a reference genome, generating a consensus sequence. The software is able to generate output files for assessment, indicating insertions, deletions and SNPs. Can handle large and complex genomes of any size.[17]
- Finally, the GS Amplicon Variant Analyzer aligns reads from amplicon samples against a reference, identifying variants (linked or not) and their frequencies. It can also be used to detect unknown and low-frequency variants. It includes graphical tools for analysis of alignments.[17]
Illumina
The technology leading to these DNA sequencers was first released by Solexa in 2006 as the Genome Analyzer.
In 2011 Illumina released a benchtop sequencer called the MiSeq. At its release the MiSeq could generate 1.5G per run with paired end 150bp reads. A sequencing run can be performed in 10 hours when using automated DNA sample preparation.[10]
The Illumina HiSeq uses two software tools to calculate the number and position of DNA clusters to assess the sequencing quality: the HiSeq control system and the real-time analyzer. These methods help to assess if nearby clusters are interfering with each other.[10]
Life Technologies
SOLiD systems was acquired by Applied Biosystems in 2006. SOLiD applies sequencing by ligation and dual base encoding. The first SOLiD system was launched in 2007, generating reading lengths of 35bp and 3G data per run. After five upgrades, the 5500xl sequencing system was released in 2010, considerably increasing read length to 85bp, improving accuracy up to 99.99% and producing 30G per 7-day run.[10]
The limited read length of the SOLiD has remained a significant shortcoming[25] and has to some extent limited its use to experiments where read length is less vital such as resequencing and transcriptome analysis and more recently ChIP-Seq and methylation experiments.[10] The DNA sample preparation time for SOLiD systems has become much quicker with the automation of sequencing library preparations such as the Tecan system.[10]
The colour space data produced by the SOLiD platform can be decoded into DNA bases for further analysis, however software that considers the original colour space information can give more accurate results. Life Technologies has released BioScope,[26] a data analysis package for resequencing, ChiP-Seq and transcriptome analysis. It uses the MaxMapper algorithm to map the colour space reads.
Beckman Coulter
Pacific Biosciences
Oxford Nanopore
MGI
MGI produces high-throughput sequencers for scientific research and clinical applications such as DNBSEQ-G50, DNBSEQ-G400, and DNBSEQ-T7, under a proprietary DNBSEQ technology.[43] It is based upon DNA nanoball sequencing and combinatorial probe anchor synthesis technologies, in which DNA nanoballs (DNBs) are loaded onto a patterned array chip via the fluidic system, and later a sequencing primer is added to the adaptor region of DNBs for hybridization. DNBSEQ-T7 can generate short reads at a very large scale—up to 60 human genomes per day.[44] DNBSEQ-T7 was used to generate 150 bp paired-end reads, sequencing 30X, to sequence the genome of SARS-CoV-2 or COVID-19 to identify the genetic variants predisposition in severe COVID-19 illness.[45] Using a novel technique the researchers from China National GeneBank sequenced PCR-free libraries on MGI's PCR-free DNBSEQ arrays to obtain for the first time a true PCR-free whole genome sequencing.[46] MGISEQ-2000 was used in single-cell RNA sequencing to study the underlying pathogenesis and recovery in COVID-19 patients, as published in Nature Medicine.[47]
Comparison
Sequencer | Ion Torrent PGM[5][49][50] | 454 GS FLX[10] | HiSeq 2000[5][10] | SOLiDv4[10] | PacBio[5][51] | Sanger 3730xl[10] | MGI DNBSEQ-G400[52] |
---|---|---|---|---|---|---|---|
Manufacturer | Ion Torrent (Life Technologies) | 454 Life Sciences (Roche) | Illumina | Applied Biosystems (Life Technologies) | Pacific Biosciences | Applied Biosystems (Life Technologies) | MGI |
Sequencing Chemistry | Ion semiconductor sequencing | Pyrosequencing | Polymerase-based sequence-by-synthesis | Ligation-based sequencing | Phospholinked fluorescent nucleotides | Dideoxy chain termination | Polymerase-based sequence-by-synthesis |
Amplification approach | Emulsion PCR | Emulsion PCR | Bridge amplification | Emulsion PCR | Single-molecule; no amplification | PCR | DNA nanoball (DNB) generation |
Data output per run | 100-200 Mb | 0.7 Gb | 600 Gb | 120 Gb | 0.5 - 1.0 Gb | 1.9~84 Kb | 1440 Gb / 1500-1800M reads |
Accuracy | 99% | 99.9% | 99.9% | 99.94% | 88.0% (>99.9999% CCS or HGAP) | 99.999% | 99.90% |
Time per run | 2 hours | 24 hours | 3–10 days | 7–14 days | 2–4 hours | 20 minutes - 3 hours | 3–5 days |
Read length | 200-400 bp | 700 bp | 100x100 bp paired end | 50x50 bp paired end | 14,000 bp ( N50 )
|
400-900 bp | 100/150/200 bp paired end |
Cost per run | US$350 | US$7,000 | US$6,000 (30x human genome) | US$4,000 | $125–300 USD | US$4 (single read/reaction) | N/A |
Cost per Mb | US$1.00 | US$10 | US$0.07 | US$0.13 | $0.13 - US$0.60 | US$2400 | $0.007 |
Cost per instrument | US$80,000 | US$500,000 | US$690,000 | US$495,000 | US$695,000 | US$95,000 | N/A |
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