Next-Generation Sequencing Applications: Diagnostics

Next-Generation Sequencing Applications: Diagnostics
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Developments in next-generation sequencing are laying the tracks for the future of diagnostics.

Next-generation sequencing is rapidly transforming applications across the scientific and medical research industry. The term "next-generation sequencing" refers to several modern sequencing techniques that researchers use to sequence base pairs in DNA and RNA samples. These techniques are key to genetic research and discovery, and, as developments in these techniques emerge, scientists are advancing treatments for genetic conditions, therapies to tackle obesity, and improved testing for COVID-19.

Furthermore, over recent years, next-generation sequencing has accelerated diagnostics in the clinical space. From minimizing the number of referrals and tests that patients must undergo to reach a diagnosis, to analyzing DNA and RNA from wastewater samples, to testing for SARS-CoV-2, next-generation sequencing has enabled vast improvements in diagnostics. Here, the peer-reviewed, open-access journal BioTechniques explains how.

How Next-Generation Sequencing Enables Precision Medicine

Scientists employ next-generation sequencing techniques in end-to-end applications that span from academic research to clinical practice, applying comprehensive genome analysis for several scientific purposes. As these techniques evolve, especially with the advent of new whole genome sequencing technologies, researchers have been able to better identify inherited disorders, characterize mutations for improved cancer diagnoses and treatments, and foresee adverse reactions to drugs and treatments.

From precision diagnostics to clinical study development, hospitals and clinics need to understand a patient's risk of disease and how they respond to new drugs and treatment protocols. Fortunately, the whole genome sequencing technology that enables this understanding is becoming standardized. This technology is now more accessible in terms of cost, speed, and the utility of captured data. As a result, precision (or personalized) medicine is now playing a major role in the research and diagnosis of cancer and rare diseases.

One of the biggest challenges associated with rare diseases is getting a diagnosis. However, as whole genome sequencing advances, it's becoming easier to diagnose patients, help them understand the causes of their symptoms, and identify treatments or other life-improving therapies. Advances in whole genome sequencing are also helping researchers understand specific genetic variants that are associated with cancer patients' tumors. This is key to developing treatments to extend patients' life expectancy and improve their quality of life.

How Next-Generation Sequencing Enables Improved Diagnostics

Sometimes, researchers use the phrase "diagnostic odyssey" to refer to the difficulty that many patients experience while seeking a diagnosis. Identifying the cause of a genetic condition can be difficult, and patients may be referred between clinicians and undergo several tests that come back inconclusive or negative. Seeking a diagnosis can take years, especially for genetic disorders, which can be among the most difficult to diagnose.

However, new technologies have made diagnosing genetic disorders much easier. Historically, researchers had to analyze data manually. Now, techniques like next-generation sequencing streamline this process and achieve more effective results. Today's advanced, faster sequencing techniques prove essential in several contexts, from diagnosing cancer to preventing long-term brain damage in children who are born with serious conditions. As technologies evolve further, it should become easier to identify treatments and therapies for rare diseases that are currently difficult to treat.

Two of the most important techniques in diagnosing rare diseases today are phenotyping (observing and recording symptoms or physical traits of a disease or condition) and genotyping (examining a patient's DNA sequence using biological assays and comparing this to a reference sequence or another individual's sequence). In future, some clinics will also combine short- and long-read sequencing so patients can receive both types of data analysis. Short-read sequencing involves the fragmentation of DNA into tiny pieces and operates to a fixed run time with bulk delivery, which can mean long wait times for results. Meanwhile, long-read sequencing examines bigger fragments of DNA and enables real-time data streaming, making this technique better-suited to time-critical applications.

Systematic Data Reanalysis

As genome sequencing advances, studies have demonstrated that reanalyzing patients' genomic sequencing data after an unsuccessful first attempt could see the number of genetic disease diagnoses rise by 4-32%. This is because when a new gene is associated with a specific condition, or when new variants in genes are reported, laboratories can re-run a patient's genomic data to determine whether the variant is present. These studies report that, despite a diagnostic yield of up to 68% (depending on the condition), many patients don't receive a diagnosis when their data is first analyzed. As a result, reanalysis could guide treatment options and improve patient outcomes

However, systematic data reanalysis isn't compulsory for laboratories. Reanalysis is time-intensive and not feasible for many laboratories to implement. Few policies address whether laboratories should reanalyze data and it has, until recently, been unclear how this has impacted clinical practice.

On top of this, a study from the Murdoch Children's Research Institute (MCRI) and the University of Melbourne has demonstrated inconsistencies surrounding decisions to reanalyze genomic data. Familial Cancer published the study, which drew on insights from 31 genetic health professionals from Europe, Canada, and Australia. These professionals established that most patients initiate reanalysis, in line with their clinicians' recommendations, after a certain amount of time has passed or upon the discovery of new genetic information. While some respondents felt that this system worked well, others challenged the system on the basis that some patients don't have the ability to request the reanalysis.

The study concluded that laboratories should initiate the reanalysis process, although the technology isn't yet developed enough for this to happen. The study also concluded that genetic services should offer clear, consistent guidelines that surround the reanalysis of genomic data. This way, patients shouldn't miss any opportunities to receive new information on their conditions.

Using Next-Generation Sequencing in COVID-19 Research

Next-generation sequencing has also been a game changer in COVID-19 research and diagnostics. In particular, the COVID-19 Genomics UK Consortium (COG-UK) has played a major role in surveying for COVID-19 and sequencing SARS-CoV-2. The consortium has used the latest techniques in next-generation sequencing to detect SARS-CoV-2 in wastewater and used the ARTIC protocol to sequence the virus.

The consortium's researchers employed PCR applications to test the samples, amplifying shorter fragments and rigorously controlling the testing environment to prevent contaminants. This way, the researchers could improve the sensitivity of the testing techniques. Whole genome sequencing has made it possible to test the wastewater samples in near real-time, instead of having to wait days to receive results. This has been essential to detecting new variants in wastewater screening.

The consortium used the ARTIC protocol, an amplicon-sequencing-based approach, to sequence SARS-CoV-2. This approach targets short amplicons from the RNA fragments (usually 200-300 base pairs), which can improve coverage for samples that are likely to be degraded (such as wastewater samples). This type of sequencing requires more experience than the alternative Midnight method but offers faster turnaround times - researchers usually receive data within 24 hours. The ARTIC protocol has developed rapidly over recent years, through three iterations, paving the way for the future of the protocol in genome sequencing.

Improving Crop Yields in East Africa With Whole Genome Sequencing

DNA sequencing and its related diagnostics have reached different stages of progression around the world. Although first-world countries have seen rapid developments in whole genome sequencing, a simultaneous lack of progression in Africa has seen many smallholder farmers living in extreme poverty. If DNA sequencing was more accessible to these farmers, they'd be able to make rapid, informed decisions about their crops and work towards reducing this poverty.

Until recently, genomic technologies that uncover complexities surrounding pests and pathogens have been unavailable in sub-Saharan Africa. These large-scale DNA sequencing machines cost over $1 million and require both large amounts of electricity and specialists to operate them. Without these machines, researchers in Africa have no choice other than to send their samples of diseased crops overseas or to the single centralized lab in Kenya. Not only is this process expensive, but the long wait for results leads to samples degrading, and it's difficult to receive the data back via weak internet connections. Sometimes, farmers wait six months for their results, by which time food shortages have already caused hunger and poverty.

To overcome this, the Tanzanian Agricultural Research Institute has delivered a molecular lab and computational resources to farmers in Tanzania, Uganda, Kenya, and the Democratic Republic of the Congo four times between 2016 and 2020. These resources make it much easier to test for cassava viruses, which destroy crop yields throughout East Africa.

Having tested the sequencing of the cassava viruses in a lab in Australia, the Institute then focused on capacity building in East Africa. This involved trialing the sequencing in labs in Tanzania, Kenya, and Uganda. Then, the Institute rolled the sequencing protocols out on the individual farms. Logistics-wise, the Institute faced challenges surrounding the shipping of temperature-dependent reagents and the lack of power and internet connection to deliver the infrastructure that the communities in East Africa so badly needed. The Institute worked closely with Oxford Nanopore to overcome these challenges, accommodating different customs, labeling, and agents. Taking this whole genome sequencing infrastructure to East Africa not only combats cassava viruses but can also handle further crop pathogen outbreaks.

Learn more from BioTechniques about how whole genome sequencing is improving access to diagnoses.

About BioTechniques

BioTechniques focuses on the laboratory methods and related technologies that are changing the face of the life sciences. Research professionals from the life sciences and disciplines like physics, chemistry, computer science, climate science, and plant and agricultural science follow the journal to stay up to date with the latest methodologies and their applications.

Since its launch in 1983, BioTechniques has grown from an independent publisher into a globally recognized name. The journal publishes reports that describe innovative new lab methods, platforms, and software; descriptions of technical tools that enable the design or performance of experiments or data analysis; surveys of technical approaches; reviews of advancements in techniques and methods; and expert opinions on experimental methods, designs, and analysis.

Aside from its journal, BioTechniques also releases podcasts, videos, and webinars on its website for its global audience. These multimedia episodes focus on topics like analytical chemistry, biochemistry, drug discovery and development, immunology, nanomedicine, and proteomics 

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