in a recent article in genome research , thompson and colleagues present a development of a next - generation sequencing technology - the heliscope sequencer - that enabled them to detect mutations in the human breast cancer 1 ( brca1 ) gene , as a model of a clinical diagnostic protocol .
it therefore holds great promise for overcoming the emerging problem in clinical genetics of target overload : the presence of a large number of gene mutations of clinical relevance in a single disease entity . in no field is this challenge more apparent than in the genomics of cancer .
the past few years have seen an unprecedented deluge of data on the genes mutated in cancer and other diseases . so far , the sequences of over 50 individual cancer genomes have been published and this number is set to increase exponentially .
the advent of next - generation technologies ( such as the roche 454 gs flx+ , llumina hiseq 2000 , applied biosystems solid and heliscope single - molecule sequencer machines ) , which allow a human genome to be sequenced in a single week - long run , has led to a large shift in our understanding of the mutations that drive cancers . for the clinician , mutations in cancer can have relevance for diagnosis , prognosis and treatment choice .
however , it is now apparent that these clinically relevant mutations will be both numerous and found at low prevalence in individual cancer types . in particular , for the majority of cancers there will be no single ' magic bullet ' targeted therapy .
the breakpoint cluster region - abelson murine leukemia homolog ( bcr - abl ) fusion kinase , which is characteristic of chronic myeloid leukemia , is the exception not the rule when it comes to druggable cancer genes ; it seems , instead , that multiple drug targets will be mutated at low frequency throughout common cancers .
this target overload is not restricted to the management of cancers and is also seen with monogenic disorders , for example , mental retardation and hereditary cancer pre - disposition .
it is therefore necessary to screen numerous genetic loci to decide on the best course of clinical management for an individual patient and this must be done in a rapid and cost - effective manner .
the current technology for searching for mutations is to amplify a region of interest - in multiple fragments of a few hundred base pairs each in separate pcr reactions - then sequence the products by standard sanger chain terminator sequencing . although highly accurate for the detection of single - nucleotide variants and small insertions or deletions , this approach is expensive , labor intensive and unable to detect large - scale insertions or deletions .
this is because sanger sequencing can reliably detect mutant alleles only when they are present in more than about 20% of the relevant dna , and this will not be the case , for example , for a heterozygous mutation in a tumor contaminated with 60% normal dna , a scenario that is not uncommon .
thompson and colleagues   have used a next - generation true single - molecule sequencing ( tsms ) system , the heliscope sequencer , to profile the mutational pattern of the human cancer gene brca1 .
germline mutations in the genes brca1 and brca2 are associated with dramatically increased rates of breast and ovarian cancers and contribute to about 10% of all breast cancer cases .
in addition , poly - adp ribose polymerase inhibitors , a recently developed family of pharmaceutical agents , seem to show selective toxicity for cancers with mutations in the brca genes . cost - effective sequencing of these genes is therefore a highly desirable clinical tool for the management of breast cancer patients and those with strong family histories of the disease .
the technology used is a development of the heliscope tsms platform . in the standard heliscope protocol
, dna is fragmented and poly(a ) adaptors are added to the ends of fragments .
they are then captured on a glass slide coated with covalently bound poly(dt ) oligonucleotides , and sequenced .
thompson et al .   were able to dispense with the initial sample preparation steps ( addition of poly(a ) adaptors and 3 ' blocking - performed to prevent extension of the 3 ' end of bound poly(a)-tagged dna molecules ) and instead directly captured only the fragments of dna belonging to the brca1 locus , using oligonucleotides that match sequences in the brca1 region ( figure 1 ) .
approximately 20% of the sequenced reads mapped to brca1 , which equates to about 100,000-fold enrichment of the target sequence . as the length of sequence that can be read in this system
is limited , oligonucleotides were designed at 20 - 30 base pair intervals throughout the coding sequence of the gene , to ensure complete coverage .
importantly , the authors   were able to obtain sequencing results from as little as 100 ng of input material - thus showing that the technology could be used with samples collected as diagnostic biopsies .
tumor material can be collected and ( a ) snap frozen to preserve intact dna and rna or ( b ) fixed in formalin then embedded in paraffin for section and histopathological review . following ( c ) dna extraction , ( d ) the dna
( e ) dna fragments are hybridized to the flow cell , which is covered with oligonucleotides with sequence complementary to the region corresponding to the brca1 gene .
( f ) each individual molecule of dna is then sequenced simultaneously by sequential addition of fluorescently labeled nucleotides ( purple ) .
this approach   has several advantages over the more traditional sanger sequencing commonly used for the detection of mutations .
firstly , there is little required in the way of sample preparation - only sonication of the dna , and in the case of archival formalin - fixed material it is possible that even this step could be dispensed with - therefore reducing cost , turn - around time and the risk of errors in sample handling . secondly ,
unlike sanger sequencing , the heliscope system allows the detection of large deletions , by determining the number of fragments of dna that are present from a specific location ; a decreased number of fragments from a region suggests a loss of genetic material .
finally , the heliscope system directly sequences individual molecules rather than - as with sanger sequencing - examining the average sequence of many millions of dna molecules .
this should provide increased sensitivity for the detection of low prevalence mutations , an essential feature in the sequencing of a heterogeneous cancer sample .
the present technology   is an advance over recently presented techniques for analyzing the mutational status of target genes using next - generation sequencing instruments . with the advent of more competitively priced next - generation sequencing machines , which can provide sequence data in a matter of hours , not days ( such as life technologies ' ion - torrent and illumina 's miseq ) , it is feasible that such approaches could be used routinely in the clinical setting .
however , the instruments involved still require the selective amplification of target dna and the relatively complex preparation of this material for sequencing .
thus , although they take advantage of the exceptionally cheap per - base cost of next - generation sequencing , competing techniques are still limited by its flaws : high complexity and slow turnaround time . genotyping by mass spectrometry has also been applied to the resequencing of relevant mutations . this approach , although sensitive , is limited , in that it is capable of detecting only known mutations and will therefore miss novel gene lesions .
for the foreseeable future , the high cost and complexity of data analysis will limit the application of whole - genome sequencing for the detection of mutations in a clinical setting .
targeted resequencing of areas of interest will therefore remain key to determining mutational status . the method published by thompson et al .  
although currently only a single gene is screened , there is clearly scope for the creation of multi - gene capture arrays , allowing large numbers of loci to be analyzed rapidly and cost - effectively with low dna input requirements . in its simplicity
, this approach provides an opportunity to truly begin integrating the vast quantity of genomic data generated in this next - generation era with clinical practice .
we would like to thank claire pike and karen howarth for helpful discussion and reading of the manuscript and the wellcome trust translational medicine and therapeutics program , breast cancer campaign and cancer research uk for funding .
 in many fields it is now desirable to sequence large panels of genes for mutation , to aid management of patients . 
 the need for extensive sample preparation means that current approaches for assessing mutation status in the clinical setting are limited . 
 a recent publication demonstrates a single - step , targeted , true single - molecule sequencing approach to assessing the mutational status of brca1 . 
 fragmented dna samples are loaded directly onto a flow cell and sequenced , thus detecting both small- and large - scale mutations with minimal sample preparation and high accuracy . 