BIOTECHNOLOGY 49
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.
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.
Tis 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).
Te 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. Te 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. Tis allows for seamless integration of performance enhancements through chemistry and software advances,” notes PacBio.
Te instrument contains three primary user access points: an RS Touch touchscreen interface. Te 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.
Troughout the DNA sequencing process, the PacBio RS uses advanced collection optics to record light pulses emitted as a by-product of nucleotide incorporation. Tese signals are delivered in real time to the primary analysis pipeline, housed entirely on the Blade Centre.
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.
Te 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.
Te MiSeq presents significant opportunities for the group’s healthcare division, applying next generation sequencing to molecular diagnostics, a key component of the business.
Te 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.
O
Application note explores the optimisation of targeted DNA sequencing However, the design of custom baits for such
xford 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.
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
www.ogt.co.uk
www.scientistlive.com
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