Issue 4 Summer 2002 - Applied Biosystems

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30 new product review Improved Software Tools for DNA Polymorphism Data Analysis D NA sequencing and polymorphic marker analysis could have been viewed as two disparate investigative techniques in the past. Direct sequencing provided a ‘gold standard’ for absolute typing of polymorphisms, allowing both detection and definition of mutations in one assay. In comparison, DNA fragment analysis provided a high-throughput, marker typing technique for analysing large numbers of samples such as the data sets needed for linkage mapping projects, or human identification databases. More recently, single nucleotide polymorphism (SNP) genotyping has bridged the gap between those original techniques, being a direct sequence interrogation assay that is applicable to mass screening projects. All these techniques face a similar challenge as genotyping projects grow in scope and complexity: the need to handle increasing volumes of data reliably and efficiently. Comparative sequencing and DNA fragment-sizing analyses, though originally undertaken by entirely separate software programmes, followed the same basic workflow, in three stages – data collection, sample file analysis and overall data review. For sequencing projects, this meant sequence ladder tracking and data collection, sequence analysis and processing by Factura and Sequence Navigator ® software. For fragment analysis the route was data collection, GeneScan ® analysis and Genotyper ® software review of the data set. One drawback of this organisation was the separation of primary data analysis and data review. All the sample data, good and bad, would have to be reviewed (and possibly edited) at the initial analysis stage before proceeding to compilation and review by the downstream software (Sequence Navigator or Genotyper software). Alternatively, analysed sample data could be loaded directly into the downstream programme and processed, but this carried the risk that poor or anomalous data from some samples would require a return to the basic analysis software for editing or re-analysis. Recently, new software products were released by <strong>Applied</strong> <strong>Biosystems</strong> which increased the efficiency of such data analysis and review, enabling much larger data sets to be handled conveniently and accurately by one operator. For comparative sequencing, SeqScape software replaces Sequence Analysis, Factura and Sequence Navigator software for reviewing data in pursuit of SNP discovery or mutation profiling. In fragment analysis and SNP projects, GeneMapper carries out all the functions formerly handled separately by the GeneScan, Genotyper and GenBase software packages. Earlier this year, updated versions of both these software programmes were released: SeqScape software v1.1 and GeneMapper software v2.0 , both with significant improvements. Both of them feature data quality indicators which enable the operator to identify immediately any sample data that needs to be reviewed, edited or possibly rejected. By eliminating the need to scan all primary data visually for acceptance, data throughput is greatly increased. Also, because review and re-analysis or rejection of a minority part of the sample data can be done within the same programme, efficiency of operation and centralising of operator intervention is much improved. As an example, an operator using GeneMapper software for a linkage project can process up to 48,000 genotypes in a six hour period, an increase of six-fold over the data-handling capacity of the previous GeneScan/Genotyper system. SeqScape software v1.1 provides Quality Values (QV) for every base call, derived from the Trace Tuner software (developed by Paracel Inc.) now embedded in the new version (see figure 1). The QV figure bears an inverse logarithmic relationship to the calculated probability of error in the base call. QV’s are displayed as scaled bars above each base call, which can be customised for colour and range by the user to allow instant identification of good, moderate and unacceptable data reliability in sequence displays. Figure 1. Quality Values This facility dramatically improves ease and speed of editing a data set to complete a project. The performance of SeqScape software v1.1 was illustrated in a poster published at the American Society for Human Genetics meeting 2001 (ref. 1). GeneMapper software v2.0 offers Process component-based Quality Values (PQV) to assist data evaluation and substantially reduce examination time (see figure 2). The PQV’s can be drawn from the many stages of sample data processing, and specific sub-sets can be selected as appropriate for certain kinds of data analysis: SNP’s or microsatellite markers, and for the latter, for dinucleotide repeats or tri- and tetra-nucleotides. Allele calls are assigned to one of three categories, pass, check and low quality, on the basis of PQV scores. A concordance control PQV allows allele calling from a known internal control sample to confirm the validity of the chosen analysis parameters for a body of related sample data. Figure 2. GeneMapper software v2.0 also features an Automatic Bin-Builder algorithm, which optimises bin centres and boundaries on the basis of recorded and processed data, to enhance reliable allele calling for micro-satellite analysis. The GeneMapper Database is pre-loaded with size standard, marker and panel information to complement our Linkage Mapping Set of human genomic markers, to help provide a complete genotyping solution. To maximise the potential of SNP based genotyping assays, GeneMapper software v2.0 has an Auto-Paneliser algorithm to allow the highest effective level of marker multiplexing when used in conjunction with the newly released SNaPshot ® Primer Focus kit. The enhanced multiplex SNP analysis capabilities of GeneMapper software v2.0 were the subject of a poster publication at ASHG 2001 (ref. 2). new product review In conclusion, ongoing and intensive research and development programmes have yielded similar benefits in throughput, accuracy and ease-of-use for both DNA sequencing and fragment analysis applications. References 1. “Enhanced performance of SeqScape software, a sequence comparison tool for variant identification” C. Kosman et al, 51st American Society for Human Genetics meeting, October 2001. 2. “Automated scoring of SNaPshot kit reactions with GeneMapper software” D. Bishop et al, 51st American Society for Human Genetics meeting, October 2001. For more information on: Improved Software Tools enter: No. 422 31
32 new product review PhotoSpray Source A New Ionisation Technique for LC/MS T he PhotoSpray source is an alternative ionisation source to TurboIonSpray ® or APCI/Heated Nebuliser for API 150EX, API 3000 and QSTAR ® Pulsar LC/MS and LC/MS/MS systems. The PhotoSpray source provides: ➜ A better sensitivity for low polar compounds ➜ A better flexibility to the ionisation range of the API 150EX, API 3000 and QSTAR Pulsar systems The availability of PhotoSpray, TurboIonSpray and APCI sources allows researchers to ionise a wider range of compounds than in the past. Principles of Photoionisation for LC/MS Photoionisation at atmospheric pressure may be used to generate ions from the vaporised LC eluent 1 . A Krypton discharge lamp (hν=10eV), can selectively ionise most analytes in the presence of the common LC solvents. Provided hν> Ionisation Potential, single photon ionisation may occur M + hν →M+ + e-. However, the efficiency of direct photoionisation is relatively poor, partly because solvent molecules and other species absorb the limited photon flux without generating ions. By adding large quantities of an ionisable dopant to the ionisation region, dopant photoions can be created in great abundance. At atmospheric pressure, in the presence of solvent and analyte, the dopant ions initiate a rapid series of ion-molecule reactions. Provided that the thermodynamics are favourable, the end result is that the charge from the dopant photoions ultimately resides with analyte molecules. M+ and/or MH+ ions are generated with high efficiency, the predominant ion type being determined by the IP and proton affinity of the analyte 2 . The PhotoSpray source in negative mode gives M-H ions with the same efficiency as APCI. Description Where the Ionspray source produces ions by the process of ion evaporation from liquid phase, the PhotoSpray source uses a Heated Nebuliser to vaporise the sample prior to inducing ionisation by atmospheric pressure photionisation. The source typically operates at temperatures between 300 and 450°C. This vaporisation process leaves the molecular constituents of the sample intact. Molecules are ionised via the process of photoionisation, induced by a beam of ultraviolet radiation in the presence of a dopant molecule, as they pass through the ion source block and into the interface region. The source block is positioned off-axis to the orifice and the optimum position is not compound dependent. Proven Curtain Gas interface technology protects the mass analyser from contamination and provides ruggedness to the system. Principles of Photoionisation for LC/MS Charge Transfer Process is suitable for non-polar compounds. This process rarely occurs with APCI. Proton Transfer Process is suitable for compounds that exhibit higher polarity. This is the dominating process. Dopant Selection The dopant is selected for its ability to undergo photoionisation, because of favourable ionisation energy – normally just below UV photon energy – and for the ease with which it can be available in high purity grade – preferably HPLC grade. Ideally it should exhibit low toxicity. Toluene, (ionisation potential of 8.83eV) meets all these requirements and is the preferred dopant compound for the PhotoSpray applications. The dopant infusion rate is 5–15% of the mobile phase flow rate (no split). Flow Rate The PhotoSpray source operates, in principle, with flow rates up to 2.0mL/min, with an optimum flow rate between 200 and 500µL/min, making the source well adapted to 2mm I.D. LC columns. Most applications have been demonstrated under this regime. The source operates under reversed or normal phase chromatographic conditions: ➜ MeOH/Water or Acetonitrile ➜ Isooctane/Isopropanol/Methylene Chloride and other LC solvents new product review PhotoSpray Applications The PhotoSpray source can ionise low polar compounds, with better sensitivity than the APCI source. PhotoSpray showed important sensitivity improvements for Steroid analysis, PolyAromatic Hydrocarbons, Vitamins, Quinones, Antioxidants, Pesticides, Pharmaceuticals and Nutraceuticals and several other classes of compounds. So the PhotoSpray source is a complementary source to the TurboIonSpray and APCI sources. TurboIonSpray Shown here is a direct plot of the relationship of PhotoIonisation to both the APCI and TurboIonSpray techniques as compound polarity increases against molecular weight. API 3000 LC/MS/MS Steroids Analysis Performance comparisons of the PhotoSpray source versus an APCI source have been performed under normal-phase chromatographic conditions for steroids 3 . Testosterone and Ethynyl Estradiol were tested with the photoionisation source and compared with the conventional APCI source. Using APCI steroids, compounds normally exhibit different sensitivity levels and fragmentation depending on their hydroxylation numbers. Ethynyl Estradiol is a steroid compound having significant economical importance for its estrogenic effect. This compound exhibits in-source decomposition and the fragment at 279amu is followed for the MRM transition. We compared the sensitivity of these steroids for the two sources in MS scan to evaluate relative ionisation efficiency and to measure LOD and LOQ using MRM transitions 4 . Toluene was used as dopant for the source at a typical flow rate of 20µL/min. A Keystone Betasil Diol 5µm, 100Å, 2x100mm has been used for the LC separation in normal phase conditions. Mobile phase composition was isocratic isooctane (94%)/isopropyl alcohol (6%). Reversed phase separations have been achieved isocratically on a BDS Hypersil Cyano 5µm, 120Å, 2 x 250mm using a mobile phase composition of MeOH (50%)/HOH (50%). Mobile phase flow rate was 200µL/min in both normal and reversed phase conditions. page 34 33
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