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Introduction

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As the ability to examine DNA has improved in resolution from chromosomal to individual nucleotide level, it has become apparent that cancer is a (usually acquired) genetic disease.1 It is therefore relevant to determine alterations in the cancer genome that bestow the ability to proliferate, prevent differentiation, defy programmed cell death and, ultimately, lead to cancer propagation.2 With the advent of Sanger sequencing in 1977, determining the nucleotide sequence in a given region of DNA became feasible. Although used for the Human Genome Project (taking 13 years and US$2.7 billion to sequence the first human genome),3 high DNA requirements, lack of sensitivity, time and cost mean widespread application of this technique to cancer sequencing is, however, impractical.

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Improvements in sequencing technology over the last two decades (collectively referred to as next generation sequencing [NGS]; Figure 6.1),4,5 mean that large parts of the germline or tumour genome can now be sequenced in a clinically relevant timeframe of hours to days. Simultaneously sequencing many genes from different patients on a single chip means costs are no longer prohibitively high and introduction of larger scale sensitive sequencing into routine clinical diagnostics is now viable. In resource-limited healthcare systems like the NHS, however, it is necessary to consider patient benefits and foreseeable pitfalls to ensure cost-effective deployment.

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Figure 6.1

Schematic representation of steps in the sequencing workflow and analysis pipeline that are common to different proprietary NGS technologies (e.g. Illumina or Ion Torrent sequencing). High molecular weight DNA requires fragmentation prior to library preparation. NGS other than whole genome sequencing (WGS), i.e. whole exome sequencing (WES) or targeted gene panels, requires selection of regions of interest for sequencing using either hybridization or amplicon (polymerase chain reaction [PCR]) approaches. Unique oligonucleotide indexes are ligated to all DNA templates from each individual patient to allow samples to be multiplexed during sequencing.

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The benefits

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Potential benefits can be divided into those relating to availability of genetic information from a patient's tumour and those relating to obtaining this information using NGS rather than conventional diagnostics.

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Genetic information may help with diagnostic uncertainty (e.g. confirming or refuting a suspected histopathological diagnosis or determining the likely origin of cancer of unknown primary), or it may provide additional prognostic information (e.g. amplification of MYCN in neuroblastoma). It is also increasingly used as a predictive tool to allow effective use of conventional treatments (e.g. withholding chemotherapy in TP53 mutated/deleted chronic lymphocytic leukaemia) or direct appropriate administration of small molecule inhibitors (according to NICE guidelines). Mutational data may also permit patients access to clinical trials, particularly of novel targeted therapeutics, while storage of genetic information in anonymized databases provides academia and pharmaceutical companies with information to direct drug development.

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Although conventional sequencing technologies could provide data for ...

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