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Next-generation sequencing technologies

Next-generation sequencing technologies are being used in clinical applications to identify circulating DNA and RNA in an effort to diagnose and define disease

Lori AS Snyder BS MA PhD
School of Life Sciences, Pharmacy, and Chemistry, Kingston University,Kingston upon Thames, UK
Next-generation sequencing provides a massively parallel, high-throughput technology to reveal the sequence of nucleic acids. Such technology has been available to us for more than 10 years, first with the availability of 454 sequencing, developed by Jonathan Rothberg and his team ( 
This technology has been followed by the introduction of the Illumia sequencing system, previously referred to as Solexa (, the ABI SOLiD system (, and the Ion Torrent sequencing systems ( In the last decade, these advances have provided rapid, cost effective, and relatively easy-to-use platforms for the sequencing of whole genomes and for selected amplicon pools. These technologies have been expanded upon, developed, and improved, enabling them to be used for research and also for experiments into their diagnostic applications.
The investigation of the diagnostic utility of circulating DNA has been pursued since it was first hypothesised that DNA could be shed from tumour cells1 in much the same way as Neisseria releases DNA into the environment.2 The diagnostic utility of identifying circulating DNA offers a promise of defining disease states and recognising the presence of disease. 
In cases where tumours are inaccessible for biopsy, where biopsy collection fails, or when the patient will not comply with invasive procedures, circulating nucleic acid detection offers an option. There is a promise that perhaps sensitive assays for circulating DNA could even identify the presence, and perhaps load, of tumours before their location in the body is identified. 
Indeed, as one example, in 1983, the concentrations of DNA in serum were used to determine that the elevated levels of circulating DNA in malignant gastrointestinal tract disease have diagnostic and prognostic value.3 In the decades since then, various methods have been applied to endeavours to develop diagnostic tests using circulating DNA. 
Until the advent of next-generation sequencing within the last decade, such explorations have been limited to targeting particular sequences using hybridisation techniques, PCR or RT-PCR. There has also been a great interest in recent years in not only investigating DNA, but also in exploring the diagnostic utility of understanding microRNAs and other small RNAs that may be circulating in the body and could represent biomarkers for disease and stages of disease progression.4
In the last few years next-generation sequencing technologies have been applied to the detection of circulating nucleic acids. By taking a sequence-based approach, the investigations are not biased by what the researchers believe should be in the sample. There is no need to design specific hybridisation probes or PCR primers for target sequences. 
All of the circulating nucleic acids can be investigated, revealing sequences that are novel and previously unexplored, as well as providing insight into the abundance and mutations within known sequences. There have been several such studies that explore the potential of these sequencing technologies; a few of the highlights indicate the range and scope in which next-generation sequencing can be applied to the investigation of circulating DNA and RNA in a variety of types of human disease.
Circulating cell-free DNA was used by Azad et al.5 to profile the tumour genomes from plasma samples that were taken from patients with metastatic castration-resistant prostate cancer. These patients were undergoing treatment for the disease. Investigators were able to observe changes in the androgen–androgen receptor gene and associate these changes with resistance to enzalutamide and abiraterone that was being experienced clinically. This study demonstrated that the DNA sequence data obtained from the 454 technology, GS FLX+ (Roche), was able to discriminate resistance gene sequences from those genotypes that were sensitive to the therapeutic drugs. 
Furthermore, because circulating DNA from plasma was used, this information could be obtained in a non-invasive manner that could monitor the genomic sequence of the tumour without requiring repeated biopsies.5 In another study using the 454 system, GS FLX (Roche), investigators analysed serum samples from females who had breast cancer and compared the results to those from females believed not to have the disease. Not only were the sequences obtained able to discriminate between individuals who did and did not have breast cancer, but discriminatory sequences were able to be identified, which categorised the stages of breast cancer development.6
Again, this information could be obtained from serum alone, without the need for repeated biopsies to monitor the progression of disease and these results may provide insight into the development of a simple to use diagnostic test.
The ability to capture and interpret circulating DNA not only can tell us about the patient, but also in the case of pregnant women, can tell us about the foetus they are carrying. Indeed, an investigation into circulating foetal DNA has revealed that it is possible to determine chromosome copy number using next-generation sequencing and therefore identify cases of trisomy 21, 18, and 13 and also cases of monosomy X.7
It was identified that the amount of circulating foetal DNA in pregnancies involving trisomy 21 was elevated, while there was a decrease in the concentration of foetal DNA when the mother was carrying a foetus with trisomy 18, trisomy 13, and monosomy X. This study confirmed the diagnostic value of the approach, using Illumina HiSeq 2000 technology, by sequencing a set of validated artificial mixtures of DNA, as well as conducting sequencing upon archived samples from established cases of these diseases.7
MicroRNAs have also been investigated on the Illumina platform, GA IIx. A study by Nimomiya et al.,8 has addressed the concern about diagnosis of primary biliary cirrhosis and was able to determine that there is a unique expression profile of microRNAs that could be identified from their data. This may provide a means to identify cases of this disease using circulating microRNA from serum.
The Applied Biosystems SOLiD version 2 next-generation sequencing platform has also been used to investigate circulating nucleic acids. A study of congenital heart defects and the utility of foetal circulating RNA to diagnose cases in utero was conducted in 2013.9 Patient samples included in the study were from mothers who were known to be carrying foetuses with one of three types of congenital heart defects: ventricular septal defect, atrial septal defect or tetralogy of Fallot. From these cases, the investigators were able to identify both up- and down-regulated microRNAs that were indicative of congenital heart defects. 
In another study using the SOLiD version 2 system, Li et al.10 investigated the presence of circulating microRNAs in women with mild and severe preeclampsia, a concern during pregnancy that can be difficult to identify rapidly and easily. From their sequence data, these researchers were able to identify differential expression in microRNAs and proposed that these could be used as diagnostic biomarkers. 
Indeed, the sequence data differentiated between women without preeclampsia, women with mild preeclampsia, and those with severe preeclampsia, showing impressive discriminatory power. It was also hypothesised that the identification of these microRNAs could reveal more about the disease itself and potential treatment options.10
The Ion Torrent Personal Genome Machine, unlike the other technologies mentioned, does not rely on reagents that have been modified to produce fluorescence. Rather, this sequencing technology uses the natural release of H+ when polymerase adds a new dNTP to the synthesising strand, to detect and determine the DNA sequence. This technology has been applied to a number of potential clinical applications, particularly as the instrumentation itself is validated and available as a diagnostic version, with dedicated diagnostic sequencing kits. 
In addition, a number of gene panel kits have been developed, which provide off-the-shelf targeted sequencing of the genes within the human genome that are believed to be of the most interest. For example, the Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion AmpliSeq Colon and Lung Cancer Panels have been used to monitor serial samples of circulating DNA in patients who are receiving tumour treatments.11 Using this data, the tumour cell genomic information was analysed to reveal changes that could be monitored during the course of a Phase I clinical trial. 
This provides a valuable tool when assessing new drugs, enabling researchers to identify mutations that may arise and potentially lead to resistance. Rothé et al. used the Ion Torrent technology to specifically investigate whether plasma could be used instead of metastatic biopsies.12 Using the Ion AmpliSeq Cancer HotSpot Panel v2, with results confirmed by Illumia sequencing, these investigators showed that plasma could be used as a viable alternative. 
This is particularly attractive in cases where there is no metastatic lesion that is accessible for biopsy, in cases where the patient is not willing to undergo an invasive procedure to obtain a biopsy, and in cases of biopsy failure. A promising study by Kurihara et al. demonstrated how the sequencing of circulating DNA using the Ion Torrent sequencing system could be used to not only detect tumours in children and identify the mutations associated with those tumours, but could also identify the presence of tumour after surgery.13
The sequence data was able to correctly identify children who had gone through surgery to have their tumours removed, yet after surgery some of the tumour still remained. In these cases, the circulating DNA specific for that tumour, containing the same mutations identified in that child’s tumour before surgery and from the removed tumour itself, was present after the surgery in cases where tumour cells remained. This can be a useful diagnostic tool to assay whether surgery has been successful in removing all of the tumour(s) from a patient.
The identification of mutations in tumours and their presence in the body is not restricted to the nucleic acids circulating in the blood. In 2015, a study was published that explored the use of cerebral spinal fluid samples to address mutations in brain tumour cells.14 As the tumours within the brain would shed material into the cerebral spinal fluid, the researchers believed this was an excellent target for circulating nucleic acid investigations when brain tumours were involved. The authors offered two options for pursuit. 
The first involved next-generation sequencing of the material within the cerebral spinal fluid. This is the preferred option when the sequence of the tumour genome is not known, as it is not biased by nor influenced by the specific targeting of certain sequences. The second option involved digital PCR and amplicon sequencing of those genes that had already been identified from that particular patient’s tumour, generally through having already obtained a biopsy or tumour tissue sample and obtaining patient-specific sequence data from the tumour itself. With the sequence already known, a targeted approach could be taken to address the sequences identified from the tumour.14
The reduction in cost, ease of use and, in some cases, diagnostic sequencing machines and dedicated kits are making the application of next-generation sequencing technologies to the investigation of circulating nucleic acids a reality. As such, we are learning more about how tumours develop, the differences in disease states as they progress, and the role of microRNAs. 
We are also identifying potential biomarkers for diseases such as preeclampsia and the stages of breast cancer, as well as developing non-invasive means to identify foetal conditions such as aneuploidies and congenital heart defects. Investigations such as those summarised here show promise that sequencing of circulating DNA and RNA will have an important role in the clinical setting.
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