RESEARCH IN PRACTICE Arch Dis Child Educ Pract Ed: first published as 10.1136/archdischild-2013-304340 on 28 August 2013. Downloaded from What is next generation sequencing?

Sam Behjati,1,2 Patrick S Tarpey1

1Cancer Genome Project, ABSTRACT deletions of DNA, large genomic dele- Wellcome Trust Sanger Institute, Next generation sequencing (NGS), massively tions of exons or whole genes and rear- Wellcome Trust Genome Campus, Hinxton, parallel or deep sequencing are related terms rangements such as inversions and Cambridgeshire, UK that describe a DNA sequencing technology translocations. Traditional Sanger sequen- 2 Department of Paediatrics, which has revolutionised genomic research. cing is restricted to the discovery of sub- University of Cambridge, Using NGS an entire human genome can be stitutions and small insertions and Cambridge, UK sequenced within a single day. In contrast, the deletions. For the remaining mutations Correspondence to previous Sanger sequencing technology, used to dedicated assays are frequently per- Dr Sam Behjati, Cancer Genome decipher the human genome, required over a formed, such as fluorescence in situ Project, Wellcome Trust Sanger Institute, Wellcome Trust decade to deliver the final draft. Although in hybridisation (FISH) for conventional Genome Campus, Hinxton, genome research NGS has mostly superseded karyotyping, or comparative genomic Cambridgeshire CB10 1SA, UK; conventional Sanger sequencing, it has not yet hybridisation (CGH) microarrays to [email protected] translated into routine clinical practice. The aim detect submicroscopic chromosomal copy Received 31 July 2013 of this article is to review the potential number changes such as microdeletions. Accepted 2 August 2013 applications of NGS in paediatrics. However, these data can also be derived Published Online First from NGS sequencing data directly, obvi- 28 August 2013 INTRODUCTION ating the need for dedicated assays while There are a number of different NGS harvesting the full spectrum of genomic platforms using different sequencing variation in a single experiment. The

technologies, a detailed discussion of only limitations reside in regions which copyright. which is beyond the scope of this article. sequence poorly or map erroneously due However, all NGS platforms perform to extreme guanine/cytosine (GC) sequencing of millions of small fragments content or repeat architecture, for of DNA in parallel. Bioinformatics ana- example, the repeat expansions under- lyses are used to piece together these lying Fragile X syndrome, or fragments by mapping the individual Huntington’s disease. reads to the human reference genome. Each of the three billion bases in the Genomes can be interrogated without bias human genome is sequenced multiple Capillary sequencing depends on pre- times, providing high depth to deliver knowledge of the gene or locus under accurate data and an insight into unex- investigation. However, NGS is com-

pected DNA variation (figure 1). NGS pletely unselective and used to interro- http://ep.bmj.com/ can be used to sequence entire genomes gate full genomes or exomes to discover or constrained to specific areas of inter- entirely novel mutations and disease est, including all 22 000 coding genes (a causing genes. In paediatrics, this could whole exome) or small numbers of indi- be exploited to unravel the genetic basis vidual genes. of unexplained syndromes. For example,

a nationwide project, Deciphering on October 1, 2021 by guest. Protected 1 POTENTIAL USES OF NGS IN CLINICAL Developmental Disorders, running at PRACTICE the Wellcome Trust Sanger Institute in Clinical genetics collaboration with NHS clinical genetics There are numerous opportunities to use services aims to unravel the genetic basis NGS in clinical practice to improve of unexplained developmental delay by Open Access sequencing affected children and their Scan to access more patient care, including: free content parents to uncover deleterious de novo NGS captures a broader spectrum of mutations than variants. Allying these molecular data Sanger sequencing with detailed clinical phenotypic infor- To cite: Behjati S, Tarpey PS. The spectrum of DNA variation in a mation has been successful in identifying Arch Dis Child Educ Pract Ed human genome comprises small base novel genes mutated in affected children 2013;98:236–238. changes (substitutions), insertions and with similar clinical features.

236 Behjati S, et al. Arch Dis Child Educ Pract Ed 2013;98:236–238. doi:10.1136/archdischild-2013-304340 Research in practice Arch Dis Child Educ Pract Ed: first published as 10.1136/archdischild-2013-304340 on 28 August 2013. Downloaded from copyright.

Figure 1 Example of next generation sequencing (NGS) raw data-BRAF V600E mutation in melanoma. The mutation was found by our group in 2002 as part of several year-long efforts to define somatic mutations in human cancer using Sanger sequencing, prior to the advent of NGS. http://ep.bmj.com/ The increased sensitivity of NGS allows detection of mosaic mutations MICROBIOLOGY Mosaic mutations are acquired as a postfertilisation The main utility of NGS in microbiology is to replace event and consequently they present at variable fre- conventional characterisation of pathogens by morph- quency within the cells and tissues of an individual. ology, staining properties and metabolic criteria with a Capillary sequencing may miss these variants as they genomic definition of pathogens. The genomes of frequently present with a subtlety which falls below pathogens define what they are, may harbour informa- on October 1, 2021 by guest. Protected the sensitivity of the technology. NGS sequencing tion about drug sensitivity and inform the relationship provides a far more sensitive read-out and can there- of different pathogens with each other which can be fore be used to identify variants which reside in just used to trace sources of infection outbreaks. The last a few per cent of the cells, including mosaic vari- recently received media attention, when NGS was used ation. In addition, the sensitivity of NGS sequencing to reveal and trace an outbreak of methicillin-resistant can be increased further, simply by increasing Staphylococcus aureus (MRSA) on a neonatal intensive sequencing depth. This has seen NGS employed for care unit in the UK.4 What was most remarkable was very sensitive investigations such as interrogating that routine microbiological surveillance did not show foetal DNA from maternal blood2 or tracking the that the cases of MRSA that occurred over several levels of tumour cells from the circulation of cancer months were related. NGS of the pathogens, however, patients.3 allowed precise characterisation of the MRSA isolates

Behjati S, et al. Arch Dis Child Educ Pract Ed 2013;98:236–238. doi:10.1136/archdischild-2013-304340 237 Research in practice Arch Dis Child Educ Pract Ed: first published as 10.1136/archdischild-2013-304340 on 28 August 2013. Downloaded from and revealed a protracted outbreak of MRSA which effective one would have to run large batches of could be traced to a single member of staff. samples which may require supra-regional centralisa- tion. Following the initial capital investment, the cap- ONCOLOGY acity of a NGS facility can provide a service on The fundamental premise of cancer genomics is that national scale likely offering economic benefits in add- cancer is caused by somatically acquired mutations, ition to improvements in patient care. and consequently it is a disease of the genome. Although capillary-based cancer sequencing has been ongoing for over a decade, these investigations were Clinical bottom line limited to relatively few samples and small numbers of candidate genes. With the advent of NGS, cancer ▸ NGS has huge potential but is presently used primar- genomes can now be systemically studied in their ily for research. entirety, an endeavour ongoing via several large scale ▸ NGS will allow paediatricians to take genetic infor- cancer genome projects around the world, including a mation to the bedside. dedicated paediatric cancer genome project.5 For the child suffering from cancer this may provide many benefits including a more precise diagnosis and classi- Funding SB receives a Wellcome Trust Clinical Fellowship. fication of the disease, more accurate prognosis, and Competing interests None. potentially the identification of ‘drug-able’ causal Provenance and peer review Commissioned; internally peer mutations. Individual cancer sequencing may, there- reviewed. fore, provide the basis of personalised cancer manage- Open access This is an Open Access article distributed in ment. Currently pilot projects are underway using accordance with the terms of the Creative Commons NGS of cancer genomes in clinical practice, mainly Attribution (CC BY 3.0) license, which permits others to distribute, remix, adapt and build upon this work, for aiming to identify mutations in tumours that can be commercial use, provided the original work is properly cited. targeted by mutation-specific drugs. See: http://creativecommons.org/licenses/by/3.0/

LIMITATIONS REFERENCES

The main disadvantage of NGS in the clinical setting 1 http://www.ddduk.org/ copyright. is putting in place the required infrastructure, such as 2 Harris SR, Cartwright EJ, Török ME, et al. Whole-genome computer capacity and storage, and also the personnel sequencing for analysis of an outbreak of meticillin-resistant expertise required to comprehensively analyse and Staphylococcus aureus: a descriptive study. Lancet Infect Dis – interpret the subsequent data. In addition, the volume 2013;13:130 6. of data needs to be managed skilfully to extract the 3 Dawson SJ, Tsui DW,Murtaza M, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med clinically important information in a clear and robust 2013;368:1199–209. interface. The actual sequencing cost of NGS is negli- 4 Chiu RW,Chan KC, Gao Y, et al. Noninvasive prenatal gible. For example, a state of the art NGS platform diagnosis of fetal chromosomal aneuploidy by massively can generate approximately 150 000 000 reads for parallel genomic sequencing of DNA in maternal plasma. around £1000 whereas a single Sanger read typically Proc Natl Acad Sci USA 2008;105:20458–63. costs less than £1. However, to make NGS cost 5 http://www.pediatriccancergenomeproject.org http://ep.bmj.com/ on October 1, 2021 by guest. Protected

238 Behjati S, et al. Arch Dis Child Educ Pract Ed 2013;98:236–238. doi:10.1136/archdischild-2013-304340