What is next generation sequencing?

Sam Behjati,1,2 Patrick S Tarpey1

1Cancer Genome Project,Wellcome Trust Sanger Institute,Wellcome Trust GenomeCampus, Hinxton,Cambridgeshire, UK2Department of Paediatrics,University of Cambridge,Cambridge, UK

Correspondence toDr Sam Behjati, Cancer GenomeProject, Wellcome Trust SangerInstitute, Wellcome TrustGenome Campus, Hinxton,Cambridgeshire CB10 1SA, UK;sam.behjati@gmail.com

Received 31 July 2013Accepted 2 August 2013Published Online First28 August 2013

To cite: Behjati S, Tarpey PS.Arch Dis Child Educ Pract Ed2013;98:236–238.

ABSTRACTNext generation sequencing (NGS), massivelyparallel or deep sequencing are related termsthat describe a DNA sequencing technologywhich has revolutionised genomic research.Using NGS an entire human genome can besequenced within a single day. In contrast, theprevious Sanger sequencing technology, used todecipher the human genome, required over adecade to deliver the final draft. Although ingenome research NGS has mostly supersededconventional Sanger sequencing, it has not yettranslated into routine clinical practice. The aimof this article is to review the potentialapplications of NGS in paediatrics.

INTRODUCTIONThere are a number of different NGSplatforms using different sequencingtechnologies, a detailed discussion ofwhich is beyond the scope of this article.However, all NGS platforms performsequencing of millions of small fragmentsof DNA in parallel. Bioinformatics ana-lyses are used to piece together thesefragments by mapping the individualreads to the human reference genome.Each of the three billion bases in thehuman genome is sequenced multipletimes, providing high depth to deliveraccurate data and an insight into unex-pected DNA variation (figure 1). NGScan be used to sequence entire genomesor constrained to specific areas of inter-est, including all 22 000 coding genes (awhole exome) or small numbers of indi-vidual genes.

POTENTIAL USES OF NGS IN CLINICALPRACTICEClinical geneticsThere are numerous opportunities to useNGS in clinical practice to improvepatient care, including:

NGS captures a broader spectrum of mutations thanSanger sequencingThe spectrum of DNA variation in ahuman genome comprises small basechanges (substitutions), insertions and

deletions of DNA, large genomic dele-tions of exons or whole genes and rear-rangements such as inversions andtranslocations. Traditional Sanger sequen-cing is restricted to the discovery of sub-stitutions and small insertions anddeletions. For the remaining mutationsdedicated assays are frequently per-formed, such as fluorescence in situhybridisation (FISH) for conventionalkaryotyping, or comparative genomichybridisation (CGH) microarrays todetect submicroscopic chromosomal copynumber changes such as microdeletions.However, these data can also be derivedfrom NGS sequencing data directly, obvi-ating the need for dedicated assays whileharvesting the full spectrum of genomicvariation in a single experiment. Theonly limitations reside in regions whichsequence poorly or map erroneously dueto extreme guanine/cytosine (GC)content or repeat architecture, forexample, the repeat expansions under-lying Fragile X syndrome, orHuntington’s disease.

Genomes can be interrogated without biasCapillary sequencing depends on pre-knowledge of the gene or locus underinvestigation. However, NGS is com-pletely unselective and used to interro-gate full genomes or exomes to discoverentirely novel mutations and diseasecausing genes. In paediatrics, this couldbe exploited to unravel the genetic basisof unexplained syndromes. For example,a nationwide project, DecipheringDevelopmental Disorders,1 running atthe Wellcome Trust Sanger Institute incollaboration with NHS clinical geneticsservices aims to unravel the genetic basisof unexplained developmental delay bysequencing affected children and theirparents to uncover deleterious de novovariants. Allying these molecular datawith detailed clinical phenotypic infor-mation has been successful in identifyingnovel genes mutated in affected childrenwith similar clinical features.

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The increased sensitivity of NGS allows detection of mosaic mutationsMosaic mutations are acquired as a postfertilisationevent and consequently they present at variable fre-quency within the cells and tissues of an individual.Capillary sequencing may miss these variants as theyfrequently present with a subtlety which falls belowthe sensitivity of the technology. NGS sequencingprovides a far more sensitive read-out and can there-fore be used to identify variants which reside in justa few per cent of the cells, including mosaic vari-ation. In addition, the sensitivity of NGS sequencingcan be increased further, simply by increasingsequencing depth. This has seen NGS employed forvery sensitive investigations such as interrogatingfoetal DNA from maternal blood2 or tracking thelevels of tumour cells from the circulation of cancerpatients.3

MICROBIOLOGYThe main utility of NGS in microbiology is to replaceconventional characterisation of pathogens by morph-ology, staining properties and metabolic criteria with agenomic definition of pathogens. The genomes ofpathogens define what they are, may harbour informa-tion about drug sensitivity and inform the relationshipof different pathogens with each other which can beused to trace sources of infection outbreaks. The lastrecently received media attention, when NGS was usedto reveal and trace an outbreak of methicillin-resistantStaphylococcus aureus (MRSA) on a neonatal intensivecare unit in the UK.4 What was most remarkable wasthat routine microbiological surveillance did not showthat the cases of MRSA that occurred over severalmonths were related. NGS of the pathogens, however,allowed precise characterisation of the MRSA isolates

Figure 1 Example of next generation sequencing (NGS) raw data-BRAF V600E mutation in melanoma. The mutation was found byour group in 2002 as part of several year-long efforts to define somatic mutations in human cancer using Sanger sequencing, prior tothe advent of NGS.

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and revealed a protracted outbreak of MRSA whichcould be traced to a single member of staff.

ONCOLOGYThe fundamental premise of cancer genomics is thatcancer is caused by somatically acquired mutations,and consequently it is a disease of the genome.Although capillary-based cancer sequencing has beenongoing for over a decade, these investigations werelimited to relatively few samples and small numbers ofcandidate genes. With the advent of NGS, cancergenomes can now be systemically studied in theirentirety, an endeavour ongoing via several large scalecancer genome projects around the world, including adedicated paediatric cancer genome project.5 For thechild suffering from cancer this may provide manybenefits including a more precise diagnosis and classi-fication of the disease, more accurate prognosis, andpotentially the identification of ‘drug-able’ causalmutations. Individual cancer sequencing may, there-fore, provide the basis of personalised cancer manage-ment. Currently pilot projects are underway usingNGS of cancer genomes in clinical practice, mainlyaiming to identify mutations in tumours that can betargeted by mutation-specific drugs.

LIMITATIONSThe main disadvantage of NGS in the clinical settingis putting in place the required infrastructure, such ascomputer capacity and storage, and also the personnelexpertise required to comprehensively analyse andinterpret the subsequent data. In addition, the volumeof data needs to be managed skilfully to extract theclinically important information in a clear and robustinterface. The actual sequencing cost of NGS is negli-gible. For example, a state of the art NGS platformcan generate approximately 150 000 000 reads foraround £1000 whereas a single Sanger read typicallycosts less than £1. However, to make NGS cost

effective one would have to run large batches ofsamples which may require supra-regional centralisa-tion. Following the initial capital investment, the cap-acity of a NGS facility can provide a service onnational scale likely offering economic benefits in add-ition to improvements in patient care.

Clinical bottom line

▸ NGS has huge potential but is presently used primar-ily for research.

▸ NGS will allow paediatricians to take genetic infor-mation to the bedside.

Funding SB receives a Wellcome Trust Clinical Fellowship.

Competing interests None.

Provenance and peer review Commissioned; internally peerreviewed.

Open access This is an Open Access article distributed inaccordance with the terms of the Creative CommonsAttribution (CC BY 3.0) license, which permits others todistribute, remix, adapt and build upon this work, forcommercial use, provided the original work is properly cited.See: http://creativecommons.org/licenses/by/3.0/

REFERENCES1 http://www.ddduk.org/2 Harris SR, Cartwright EJ, Török ME, et al. Whole-genome

sequencing for analysis of an outbreak of meticillin-resistantStaphylococcus aureus: a descriptive study. Lancet Infect Dis2013;13:130–6.

3 Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of circulatingtumor DNA to monitor metastatic breast cancer. N Engl J Med2013;368:1199–209.

4 Chiu RW, Chan KC, Gao Y, et al. Noninvasive prenataldiagnosis of fetal chromosomal aneuploidy by massivelyparallel genomic sequencing of DNA in maternal plasma.Proc Natl Acad Sci USA 2008;105:20458–63.

5 http://www.pediatriccancergenomeproject.org

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