Table 1 gives a summary of the technical specifications of each of these instruments. Here we evaluate the output of these new sequencing platforms and compare them with the data obtained from the Illumina HiSeq and GAIIx platforms. The Illumina Genome Analyzer and more recently the HiSeq 2000 have set the standard for high throughput massively parallel sequencing, but in 2011 Illumina released a lower throughput fast-turnaround instrument, the MiSeq, aimed at smaller laboratories and the clinical diagnostic market. In recent years, the sequencing industry has been dominated by Illumina, who have adopted a sequencing-by-synthesis approach, utilizing fluorescently labeled reversible-terminator nucleotides, on clonally amplified DNA templates immobilized to an acrylamide coating on the surface of a glass flowcell. As each base is incorporated, a distinctive pulse of fluorescence is detected in real time. The width of the ZMW is such that light cannot propagate through the waveguide, but energy can penetrate a short distance and excite the fluorophores attached to those nucleotides that are in the vicinity of the polymerase at the bottom of the well. Each polymerase is allowed to carry out second strand DNA synthesis in the presence of γ-phosphate fluorescently labeled nucleotides. Here, DNA polymerase molecules, bound to a DNA template, are attached to the bottom of 50 nm-wide wells termed zero-mode waveguides (ZMWs). PacBio have developed a process enabling single molecule real time (SMRT) sequencing. If bases of that type are incorporated, protons are released and a signal is detected proportional to the number of bases incorporated. As sequencing proceeds, each of the four bases is introduced sequentially. The templated beads are loaded into proton-sensing wells that are fabricated on a silicon wafer and sequencing is primed from a specific location in the adapter sequence. DNA fragments with specific adapter sequences are linked to and then clonally amplified by emulsion PCR on the surface of 3-micron diameter beads, known as Ion Sphere Particles. #Final draft tagger torrent torrentThe Ion Torrent PGM “harnesses the power of semiconductor technology” detecting the protons released as nucleotides are incorporated during synthesis. Of note were the Ion Torrent Personal Genome Machine (PGM) and the Pacific Biosciences (PacBio) RS that are based on revolutionary new technologies. Sequencing technology is evolving rapidly and during the course of 2011 several new sequencing platforms were released. However there are key differences between the quality of that data and the applications it will support. ConclusionsĪll three fast turnaround sequencers evaluated here were able to generate usable sequence. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Together, these represent a comprehensive range of genome content. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Next generation sequencing (NGS) technology has revolutionized genomic and genetic research.
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