New DNA sequencers offer biology in real-time

The market for DNA sequencers is growing, both in the face of demand and in line with the emergence of new, faster technologies. Here Sean Ottewell looks at the latest releases.

The market for DNA sequencers increased in the last year with the introduction of new systems and a growing demand for sequencing, according to Kalorama Information.

The healthcare market research publisher said that the introductions of the Ion Torrent PGM, PacBio RS and MiSeq grew the sequencer market nearly 5 per cent to US$560m (EUR429m) in 2011. More new technologies are expected from Ion Torrent as well as from Oxford Nanopore that may further change the market and introduce price changes, according to Kalorama's report, 'DNA sequencing equipment and services markets, 3rd edition'.

"This was getting to be a stable market, but that changed in the last two years," said Justin Saeks, Kalorama analyst and author of the report. "Now it's more volatile and complicated; we think customers will acquire new systems, but pricing and feature preferences will get interesting."[Page Break]

According to the report, DNA sequencing has seen very high growth in recent years as a result of labs deciding to start using next-generation sequencing for their research. At the same time, large segments of end-users are still continuing to use CE Sanger systems and may take a while to shift to the new technologies.

The science of sequencing is constantly evolving, as more clinical applications are sought and competition with other technologies is resolved. Just one of many trends is that microarrays remain more cost-effective for genome-wide association studies, but given the trends in the science and sequencing costs, that could change too, creating a further boost for DNA sequencers.[Page Break]

Another trend is that large structural variations have shown to be highly important for genetic variation, more so than single nucleotide polymorphisms (SNPs); read lengths then become a key factor in system purchases. As specific applications place greater emphasis on one benefit or another, the market segments can quickly narrow down their usage to certain products.

"Longer-term, questions still remain for broad areas such as disease prevention and diagnostics which could turn into home runs or strikeouts," Saeks said.[Page Break]

The new technologies

According to Life Technologies, researchers can now have it all - affordability and full flexibility to perform sequencing of small whole genomes, sets of genes, or transcripts - all on the Ion PGM system.

Different sequencing applications have demands, so in addition to the highly accurate Ion PGM 200 Sequencing Kit, the company has introduced the Ion PGM Sequencing 300 Kit powered with a new long-read sequencing enzyme. Life Technologies says it is committed to further expanding long read sequencing capabilities and expects to support 400-base sequencing by the end of 2012 using the same long-read sequencing chemistry that is in the 300-base kit.

The Ion PGM Sequencing 300 Kit is designed for de novo and long-amplicon sequencing applications such as HLA and 16S rRNA typing. Analysis from 200-base and 300-base sequencing runs generated at Ion Torrent demonstrate higher quality assemblies for 300-base sequencing, with increases in N50 values of 50 per cent and 150 per cent for E. coli DH10b and S. aureus, respectively, and dramatic increases in the size of the largest contig of 67 per cent and 97 per cent for each organism, respectively.[Page Break]

In addition to de novo sequencing, some targeted sequencing applications including HLA and 16S rRNA typing benefit from longer reads, as the key target regions are long contiguous stretches that span large exons or contain multiple adjacent hypervariable regions. Additionally, RNA sequencing projects that aim to discover and detect isoforms and novel transcripts are better enabled with long reads that span exon-exon junctions. Performance results from in-house runs demonstrate that the Ion PGM Sequencing 300 Kit consistently yields individual reads with long read lengths and high run throughput.

For targeted gene sequencing applications where variant detection is critical and formalin-fixed, paraffin-embedded (FFPE) samples are often used, Life Technologies offers the Ion PGM 200 Sequencing Kit in combination with Ion AmpliSeq Ready-to-use Panels or custom panels designed by Ion AmpliSeq Designer, Torrent Suite Software v3.0, and Ion Reporter Software.[Page Break]

This end-to-end product solution delivers a robust gene panels that require only 10ng of DNA per primer pool and the sequencing chemistry that is optimised for high raw accuracy. Variant calling can be performed by Torrent Suite Software v3.0 or Ion Reporter Software. Both software tools are equipped with new and improved variant calling algorithms, enabling accurate detection of SNPs and additionally indels within large homopolymer regions.

Meanwhile the PacBio RS is a third generation DNA sequencing system that incorporates novel, single molecule sequencing techniques and advanced analytics to reveal true biology in real time.

Called single molecule, real-time (SMRT) technology, the system conducts, monitors and analyses biochemical reactions at the individual molecule level, according to users' specific protocols and experiment designs. Long readlengths, intuitive operation and throughput flexibility combine to deliver the required data at faster speeds than previously possible (Fig.1).[Page Break]

The instrument features high performance optics, automated liquid handling, and an environmental control centre, all directed through an intuitive touchscreen interface. Also included is a state-of-the-art Blade Centre, the computational brain responsible for primary data analysis. The comprehensive informatics suite, for more advanced analyses, completes the package.

"Because we are continuously working to expand our capabilities, the instrument is designed with maximum scalability. This allows for seamless integration of performance enhancements through chemistry and software advances," notes PacBio.

The instrument contains three primary user access points: an RS Touch touchscreen interface. The system's primary control centre, this features an adjustable viewing angle and is fully compatible with gloved use; a reagent and sample drawer for plate loading; and SMRT cell and tip drawer for consumables loading.

Throughout the DNA sequencing process, the PacBio RS uses advanced collection optics to record light pulses emitted as a by-product of nucleotide incorporation. These signals are delivered in real time to the primary analysis pipeline, housed entirely on the Blade Centre.[Page Break]

Proprietary algorithms translate each pulse into an A, C, G or T base call with its own set of quality metrics. As soon as the basecall data is generated, it is available for secondary analysis through PacBio software or virtually any other secondary analysis pipeline.

Additional features of the PacBio RS include: intuitive run setup tools; workflow optimisation tools; error-proof instrument loading; robotic workflow management; absolute run size flexibility; and intelligent run progress monitoring.

Finally, Source BioScience has launched an extended next generation sequencing service using the new Illumina MiSeq technology platform.

The MiSeq service addresses the middle ground between the group's existing overnight service for sequencing and the ultra high throughput HiSeq service, enabling access to new markets and customers for next generation sequencing.

The MiSeq presents significant opportunities for the group's healthcare division, applying next generation sequencing to molecular diagnostics, a key component of the business.[Page Break]

The latest developments in cancer diagnostics and personalised treatment for patients are aimed at creating integrated genetic tests that characterise cancers at the molecular level to improve diagnosis and treatment outcome.

Source BioScience is the only MiSeq service provider in the UK and a CSPro certified Illumina partner. With the MiSeq Personal Sequencing System, the company guarantees rapid - 24 hour - turnaround times for DNA samples.[Page Break]

Application note explores the optimisation of targeted DNA sequencing

Oxford Gene Technology (OGT), provider of innovative clinical genetics and diagnostic solutions to advance molecular medicine, has released a new application note exploring the optimisation of targeted DNA sequencing.

Entitled 'Fishing for variants in the deep end of the gene pool', the document discusses how the intelligent design of custom bait probes can significantly improve data quality by decreasing off-target noise, increasing the depth of coverage and optimising the sensitivity of variant detection. Well-designed probes can also be used to investigate GC-rich areas of the genome, thereby ensuring studies are not biased towards low-GC regions.

Whilst whole exome sequencing offers a powerful route into analysis of Mendelian disorders and provides a platform for genome wide association (GWAS) studies, custom designs offer significant advantages where the biological question is more focused.

Examples include the follow up of GWAS, or investigations into the mutational analysis of specific pathways or genes in a clinical context. Targeted sequencing offers greater depth of coverage, reduced cost, simpler data analysis and shorter time to results.

However, the design of custom baits for such studies is not straightforward and poor design strategies can render the results unusable. For this reason, through its Genefficiency targeted sequencing services, OGT leverages its expertise in probe design for solid and liquid phase hybridisation to create custom bait probes optimised for each individual study, ensuring accurate, unbiased results that provide true biological insight.

To find out more, and to download the new application note, visit

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