One of the joys of science is posing and investigating big questions. Scientists pose and re-pose, digging deeper each time to detect a clear answer in a complex world. Researchers studying rare diseases ask how newly arising pathogenic mutations can be identified. Elsewhere, scientists try to understand how individual cells work within a complex system such as our neurological system to understand the connectivity of the network.

But what if your technology of choice blocks you from certainty in the answer you seek? Sequencing technology has become increasingly powerful over the last few years with many new platforms that boast improved cost per sample, depth of reads, and throughputs. With the many sequencing options now available, choosing the right one can be challenging. Should you choose the technology that is referenced most often in research papers? How do you know it's the right choice to answer your question?

Because that is root of the decision-making process: your questions and what you are trying to accomplish. Are you trying to identify which factors cause a mutation? Isolate a known variant? Verify an unexpected discovery? Once you have articulated your question, you can determine the best technology or combination of technologies to obtain an answer with a high degree of certainty.

Let’s look at a few types of questions that scientists are asking and how the various sequencing technologies can best answer them.

Identification of clinically relevant cancer mutations

Clinical researchers studying colorectal cancer typically query certain hotspot mutations of the KRAS, NRAS, and BRAF genes in order to choose an appropriate therapy and assess drug response. This implies that the target regions are defined and variants contained within those regions well-characterized.  In these cases, querying a panel of defined target regions would be suitable to find variants. The question then is: which sequencing approach is preferred? Targeted NGS or Sanger sequencing?  For routine screening, where low cost and an easy workflow is needed, Sanger sequencing may be preferable.  Moreover, Sanger sequencing can comfortably detect variants that make up at least 5% of the alleles in a sample.  Alternatively, targeted NGS panels offer greater flexibility for accommodating larger numbers of targets or larger numbers of samples per sequencing run.  And when tumor content is low, as in the case of liquid biopsies, NGS can provide greater sensitivity for detecting variants as low as 0.1% of the total in a sample.

Identification of mutations for genetic diseases

Certain disorders show clear heritable contributions with classical single-gene Mendelian-inherited patterns of transmission.  Other syndromes, such as stroke, demonstrate a clear heritability, but cannot be traced to a single causative locus.  Sequencing panels of candidate genes, together with pedigree analysis, can help identify causative loci and understand familial susceptibility to a syndrome.  Sanger sequencing panels can be cost effective when the number of loci or samples are low; NGS panels may be preferable for a large target number or large number of samples.  Finally, if there is no hypothesized candidate locus or loci for the disorder, whole exome or whole genome NGS of members in a pedigree might uncover the causative mutation (or mutations).

Analyzing bacteria that contribute to infection

Pathogenic bacterial infections are a major contributor to poor human health.  Sometimes it can be challenging to determine a corrective course of action due to the difficulty in accurately ascertaining which bacteria species are present.  For example, doctors studying complications from diabetes struggled to prescribe the correct antibiotic for their patients suffering from diabetic foot ulcers.  If a single organism is suspected, its identity can be determined by Sanger sequencing the 16S rRNA gene and querying a database.  On the other hand, targeted NGS sequencing of a panel of bacterial species allows scientists to analyze what could be dozens of bacteria species present.  If the identity of the of the species is not known, an unbiased NGS approach, where the entire genome of the unknown organism (or organisms) is determined, may be useful in identifying what is present in the sample.

Understanding conditions that have no clear cause

In some rare diseases, scientists may be unsure which part of the genome should be the focus of sequencing efforts. For patients with congenital anomalies and intellectual disability, their condition often goes undiagnosed or misdiagnosed even after a long and stressful series of diagnostic tests (1). Because unbiased WGS or WES are useful technologies for broad discovery questions, they can serve as comprehensive genomic tests to capture nearly all the genomic variation and point to the causative molecular lesion. Targeted NGS would not work as well for these types of studies, as its hypothesis-driven approach implies that the scientist has some knowledge beforehand about what type of genetic change may be responsible.

Understanding how a virus spreads

Targeted NGS panels have been widely used for outbreak investigation and identification. Their ability to simultaneously analyze a large number of targets makes them ideal to understand how pathogens interact with their hosts, how they relate to each other, and how drug resistance genes spread within a community. Researchers in West Africa trying to understand the Ebola virus needed the power of NGS technology to quickly understand the epidemiology and evolution of the virus.

These are just a few of the types of questions that can be answered with sequencing technologies. Whether your questions are in oncology, inherited diseases, infectious diseases, or other fields of research, there are a variety of technologies that can give you the analytic certainty you need to support your discovery, develop the best therapeutic approach, or build a database for future diagnoses. All of these outcomes are dependent on you continuing to ask big questions. Keep asking.

See related article for a deeper perspective on sequencing approaches for genetic disease research.

1. Nambot S, Thevenon J, Kuentz P, Duffourd Y, Tisserant E, Bruel AL, Mosca-Boidron AL, Masurel-Paulet A, Lehalle D, Jean-Marçais N, Lefebvre M, Vabres P, El Chehadeh-Djebbar S, Philippe C, Tran Mau-Them F, St-Onge J, Jouan T, Chevarin M, Poé C, Carmignac V, Vitobello A, Callier P, Rivière JB, Faivre L, Thauvin-Robinet C; Orphanomix Physicians' Group. Clinical whole-exome sequencing for the diagnosis of rare disorders with congenital anomalies and/or intellectual disability: substantial interest of prospective annual reanalysis. Genet Med. 2017 Nov 2. [Epub ahead of print] PubMed PMID: 29095811.

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