Next Generation Sequencing: A Disruptive Genome Analysis Technology

Next Generation Sequencing: A Disruptive Genome Analysis Technology

Next Generation Sequencing: Disruptive Genome Analysis Technology

The first consensus sequence of the human genome in 2001 took more than a decade to complete and cost an estimated $70 million dollars.1 It utilized primarily sequencing technology first introduced by Sanger in 1977.

Even though the Sanger method, the gold standard, is the most accurate and widely available sequencing method, it has a low throughput and is capable of reading DNA fragments of about 1 kb in length. It has been estimated that on one machine, using Sanger sequencing to sequence the entire human genome would cost $5-$30 million and take about 60 years.2

Next Generation Sequencing (NGS) solves these issues by allowing massively parallel sequencing of the genome to obtain high output (up to millions of DNA fragments) on each run. It costs substantially less than Sanger sequencing and has emerged as a revolutionary genomic tool. This molecular microscope is not only decoding groups of related genes, exomes, and whole genomes, but also a variety of processes including replication, transcription, translation, methylation, and nuclear DNA folding.



Since its advent in 2005, NGS has demonstrated its enormous potential in clinical medicine, diagnostics, personalized medicine, and life sciences research. The practical applications of these technologies are disrupting the research and clinical communities and leading the way toward better and faster health care.


NGS Applications

    • Sequencing Cancer Genes: Cancer research has traditionally been complicated because different types of cancers do not share common underlying mechanisms. Scientists have to study a multitude of genetic variations that may lead to different cancer phenotypes, and they must compare sequences between cancer patients and healthy individuals to detect which variations are implicated in tumorigenesis. Unveiling and comparing these variations in a sufficient number of individuals cannot be done quickly or cost-effectively by traditional sequencing methods. However, with NGS, many genomes can be sequenced simultaneously, which has led to commercially available gene panels for cancer screening, diagnosis, prognosis, and pharmacogenesis. For instances, the Extended RAS Panel, an FDA-approved NGS kit, helps clinicians identify colorectal cancer patients eligible for Panitumumab treatment. NGS is encouraging worldwide collaborations for cataloging the mutations and genomic signatures in target genes for risk stratification, outcome prediction, and new treatment paradigms.
    • Sequencing Whole Exomes: NGS technologies can selectively sequence the whole exome (protein coding regions) to discover most mutations that cause common and rare human diseases. While these coding regions constitute only 1-2% of the human genome, they contain about 85% of all disease-causing mutations.
    • Sequencing Cell-Free DNA: NGS provides an efficient method to detect, measure, and annotate cell-free nucleic acids circulating in human blood. These can be used as early molecular diagnostic markers for many diseases, such as diabetes, cancer, and myocardial infarction. Identification of tumor- and fetal-derived circulating DNA has also been studied extensively for clinical oncology and prenatal diagnosis.
    • Gene Expression Profiles: NGS can decipher the presence and quantity of RNA in a biological sample through RNA sequencing (RNA Seq) more accurately than microarrays. It can analyze the continuously changing cellular transcriptome by looking at alternatively spliced gene transcripts, post-transcriptional modifications, gene fusions, mutations/SNPs, and changes in gene expression over time to study the efficacy of various treatment options.
    • Personal Genome: Whole-genome sequencing can yield useful and clinically relevant information for individual patients and, with the cost of sequencing falling precipitously, the widespread use of personal genomes appears within reach. The next challenge will be deciphering relevant information from large volumes of data from individuals for personalized medicine. Addressing this challenge will require comprehensive databases for disease-specific mutations so that proper diagnoses can be made and the proper treatments selected.
    • New Disease Genes/Genetic Disorders: NGS has been successfully applied in many different research studies to identify new disease genes and genetic disorders. These discoveries include single gene disorders such as neurofibromatosis type 1, Marfan syndrome, and spastic paraplegia, as well as diseases caused by a group of related genes such as hypertrophic cardiomyopathy and congenital disorders of glycosylation. NGS has also been applied to multi-gene disorders, including retinitis pigmentosa and disorders without identified genetic causes
    • Diagnosing Polygenic Diseases: For monogenic diseases with clear clinical and biochemical presentations, Sanger sequencing is an accurate and cost-effective way to obtain a conclusive molecular diagnosis. Because most inherited diseases are genetically heterogeneous, sequence analysis is costly and time consuming, which delays diagnosis and treatment. For example, 65 genes are responsible for Retinitis Pigmentosa, which renders traditional sequencing methods inadequate for analysis.

    In the context of clinical applications, NGS technologies offer a number of advantages over the traditional sequencing methods. Specifically, NGS allows sequencing of a group of biomarkers from multiple samples in each run to get faster results for patients. Each patient can also be simultaneously screened for various genomic aberrations such as single nucleotide and multi-nucleotide polymorphisms (SNPs), insertions and deletions (indels), copy number variants (CNVs), and gene/transcript fusions. Simultaneous screening enables clinicians to conduct multiple tests in one run, lowers healthcare costs, and reduces patient sample requirements

    • Pre-natal Diagnosis: Traditionally, prenatal diagnosis requires invasive methods like amniocentesis or chorionic villus sampling to detect chromosomal abnormalities. Besides cost, these procedures pose a miscarriage risk at an approximate rate of 0.5%. Therefore, it is highly desirable to develop a non-invasive method for prenatal diagnosis to avoid the risk of fetal loss. One of the most valuable applications of NGS technologies is molecular genetic testing in pre-diagnostics. There has been rapid progress in applying NGS technologies to the detection of fetal chromosomal abnormalities in fetal DNA from cell-free DNA fragments in maternal plasma.

      These wide-ranging applications of NGS are barely scratching the surface. As the technology matures, it has the promise to revolutionize molecular diagnostics, personalized medicine, and scientific research.


      We at Arc Bio have recently introduced GalileoTM AMR, the most advanced gram-negative plasmid AMR detection and annotation software. This best-in-kind AMR knowledgebase contains the most extensive archive of expert-validated gram-negative AMR genes, cassettes, and other mobile elements.

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      1. Venter, J. C., et al. The sequence of the human genome. Science. 2001; 291: 1304–1351.
      2. Wong K. Computational Biology and Bioinformatics: Gene Regulation. CRC Press. April 27, 2016.
      3. Nguyen HT, Le HTT, Nguyen LT, Lou H, LaFramboise T. The applications of massive parallel sequencing (next-generation sequencing) in research and molecular diagnosis of human genetic diseases. Vietnam Journal of Science, Technology, and Engineering. 2018; 60(2): 30-43.

      Alarming Increase of Antimicrobial Resistance: Prioritization of Bacterial Targets for Research by the WHO

      Alarming Increase of Antimicrobial Resistance: Prioritization of Bacterial Targets for Research by the WHO

      Alarming Increase of Antimicrobial Resistance: Prioritization of Bacterial Targets for Research by the WHO

      According to the World Health Organization (WHO), antimicrobial resistance (AMR) is a growing threat to global public health undermining effective prevention and treatment of disease caused by bacteria, parasites, viruses, and fungi.1

      Without effective antibiotics:

      • Major surgeries such as caesarean section, organ transplants, hip replacements, and chemotherapy become riskier
      • More tests and use of costlier drugs are needed
      • Illnesses are prolonged which may result in worse clinical outcomes
      • The fight against HIV, TB, and malaria is complicated

      The rate of AMR is being accelerated by the misuse and overuse of antibiotics in humans, utilization as growth promoters, or to prevent diseases in healthy animals. Poor infection control, unsanitary conditions, and improper handling of food also contribute to the spread of AMR.

       Acquired Resistance in Specific Bacteria

      Klebsiella pneumoniae is a major cause of hospital-acquired infections such as pneumonia, bloodstream infections, infections in newborns, and in intensive care unit (ICU) patients. This common intestinal bacterium has developed resistance to a class of antibiotics, carbapenems, throughout the world.

      Escherichia coli is another common intestinal bacterium that has developed widespread resistance to fluoroquinolone antibiotics used to treat urinary tract infections

      Cephalosporins are last line of defense against gonorrhea but bacterial resistance has been confirmed in several countries including Australia, Austria, Canada, France, Japan, Norway, Slovenia, South Africa, Sweden, U.K, and Northern Ireland. Such growing resistance has prompted the WHO to update treatment guidelines for gonorrhea, syphilis, and chlamydial infections.

      Resistance to first-line drugs to Staphylococcus aureus, a common cause of severe infections in health facilities and the community, is widespread. Mortality rates of people with methicillin-resistant S. aureus (MRSA) is 64% higher than people with a non-resistant form of the infection.

      Colistin is the treatment of last resort for life-threatening infections caused by Enterobacteriaceae which are already resistant to carbapenems. Resistance to colistin has been detected in several countries, making infections untreatable.

      WHO estimates that, in 2014, there were about 480,000 new cases of multidrug-resistant tuberculosis (MDR-TB), a form of tuberculosis that is resistant to the two most powerful anti-TB drugs. Extensively drug-resistant tuberculosis (XDR-TB), which is resistant to at least four anti-TB drugs, has been identified in 105 countries.

      As of July 2016, resistance to the first-line treatment for Plasmodium falciparum malaria has been confirmed in 5 countries (Cambodia, the Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam). Along the Cambodia-Thailand border, P. falciparum has become resistant to almost all available antimalarial medicines.

      To protect public health, there is a need for coordinated action between countries, scientists, and governmental bodies. WHO is providing technical assistance to help countries develop their national action plans and strengthen their health and surveillance systems to more effectively prevent and manage antimicrobial resistance. WHO has established multiple initiatives to address antimicrobial resistance, e.g., World Antibiotic Awareness Week has been held every November since 2015.

      Member states have asked the WHO to identify the most important resistant bacteria globally in terms of urgency for new treatments and to guide prioritization of incentives and funding for public health needs.2 Mycobacterium tuberculosis was not discussed because its urgent need has previously been established. Criteria for prioritization included all-cause mortality, healthcare and community burden, prevalence and 10-year trend of resistance, transmissibility, preventability, treatability, and current pipeline.

      A group of 70 experts stratified the results in three priority tiers: critical, high, and medium.

      Priority 1: CRITICAL

      • Acinetobacter baumannii, carbapenem-resistant (gram-negative)
      • Pseudomonas aeruginosa, carbapenem-resistant (gram-negative)
      • Enterobacteriaceae which include K. pneumonia, E. coli, Enterobacter spp., Serratia spp., Proteus spp.,
      • Providencia spp., Morganella spp., carbapenem-resistant, 3rd generation cephalosporin-resistant (gram-negative)

      Priority 2: HIGH

      • Enterococcus faecium, vancomycin-resistant (gram-positive)
      • S aureus, methicillin-resistant, vancomycin intermediate and resistant (gram-positive)
      • Helicobacter pylori, clarithromycin-resistant (gram-negative)
      • Campylobacter spp., fluoroquinolone-resistant (gram-negative)
      • Salmonella spp., fluoroquinolone-resistant (gram-negative)
      • Neisseria gonorrhoeae, 3rd generation cephalosporin-resistant, fluoroquinolone-resistant (gram-negative)

      Priority 3: MEDIUM

      • Streptococcus pneumoniae, penicillin-non-susceptible (gram-positive)
      • Haemophilus influenzae, ampicillin-resistant (gram-positive)
      • Shigella spp., fluoroquinolone-resistant (gram-negative)
      The list shows gram-negative bacteria (top 3) as a critical priority. It is vital for the scientific community to continue improving understanding of mechanisms bacteria use to acquire resistance and to develop new antibiotics against multidrug- and extensively drug-resistant pathogens.


      We at Arc Bio have recently introduced GalileoTM AMR, the most advanced gram-negative plasmid AMR detection and annotation software. This best-in-kind AMR knowledgebase contains the most extensive archive of expert-validated gram-negative AMR genes, cassettes, and other mobile elements to support researchers targeting bacteria prioritized by the WHO.

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      1. WHO guidelines on use of medically important antimicrobials in food-producing animals. 2017.
      2. Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. World Health Organization. 2017. ET_NM_WHO.pdf