The State of Next Generation Sequencing, in a Glimpse

The State of Next Generation Sequencing, in a Glimpse

Next-generation Sequencing (NGS) refers to non-Sanger-based high-throughput DNA sequencing tech. A significantly greater throughput can be achieved through NGS, through the sequencing of millions or even billions of strands of nucleic acids in parallel, without so much need for the fragment-cloning methods used in Sanger. While Sanger-based sequencing, with 99.99% accuracy, is the gold standard in genome sequencing, next-generation sequencing is making strides in the clinical laboratory with some major advantages.

Sanger sequencing, performed on a gel, produces one long forward/reverse read—700 base pairs per sequence—but only yields about 70 kilobases per hour. Meanwhile, when running two flow cells on the NovaSeq, Illumina’s flagship NGS platform, one could produce up to 6,000 gigabases (that’s six billion kilobases) in 20 billion reads per 44-hour run with read lengths of up to 300 bases from both ends of the template. Turnaround time for a typical read with NGS is about four hours. The cost of NGS is substantially lower as well, with fifty cents per kilobase, as opposed to Sanger’s $500 for the same.

Each has their place; if a lab needs low-cost, high-throughput sequencing for over a hundred genes at a time, and/or have little sample material with which to work, next-generation sequencing is the way to go. NGS is also better when it comes to finding novel gene variants through expanded targeting, and for sequencing microbial genomes for pathogen subtyping during a critical outbreak.

This is not to say Sanger is outmoded: while it is slow and low-yield relative to NGS, not to mention a great deal more costly at the kilobase level, its accuracy is unmatched. If you are targeting fewer genes, fragments, or even sequencing a single gene, then with its near infallibility, Sanger is the answer. It is also best for analyzing single tandem repeats and identifying microbes outside of outbreak scenarios. And ultimately, Sanger-based sequencing is often used in confirmation of NGS results.

Below is a selection of companies involved in NGS in some form or another, whether in the development of NGS platforms, applications in sequencing, or through providing services that rely on NGS. There are plenty more, and we offer profiles on these companies and more in Kalorama’s wide range of reports. The eleventh edition of our report, The Worldwide Market forIn VitroDiagnostic (IVD) Tests, is coming this summer, and Next Generation Sequencing Markets (Instrumentation, Consumables, Services, Competitive Analysis, Trends) was released in April.

ARUP Laboratories began offering high-resolution NGS-based human leukocyte antigen (HLA) genotyping under an agreement with The Children’s Hospital of Philadelphia (CHOP) in November 2014, through two NGS HLA tests, HLA Class I (A, B, and C) and HLA Class II (DRB1 and DQB1).

In March 2018, ARUP and metagenomics company IDbyDNA, Inc. launched Explify Respiratory, an NGS test for respiratory infections that assists physicians having difficulty in diagnosing patients with pneumonia and other respiratory diseases. The test uses nucleic acid analysis to identify over 200 common and rare bacteria, viruses, fungi, and parasites with a single test, helping to reduce unnecessary antibiotic use and sequential testing, and to shorten hospital stays. The test identifies organisms by their genetic material through the use of IDbyDNA’s Taxonomer DNA search engine software.

In January 2018, ARUP launched a new bioinformatics pipeline and cloud-based computing infrastructure for NGS testing, called Pipey. To meet the increasing demand that the company had been recently facing as costs decrease, the platform was designed to simultaneously enable more efficient processing for large sample volumes and support shorter turnaround times; everything, from software, to hardware, to infrastructure, was rebuilt from the ground up. Users identify clinically significant mutations, while Pipey takes raw sequencing data and generates lists of genetic variants of interest with associated annotations using variant calling algorithms. Using cloud-based computational power, 10,000 samples can be run in the same span of time as a single sample.

GeneDx is BioReference Laboratories' genetics laboratory and concentrates on testing of rare genetic diseases. GeneDx offers GenomeDx, based on a CGH (comparative genomic hybridization) array platform and its next gen sequence offerings that are disease specific. BioReference estimates that GeneDx offers the largest ultra-rare genetic tests in the industry. It marketed its first molecular microarray in 2007 and launched next generation sequencing in 2008. BioReference reported that GeneDx was the first commercial laboratory to utilize next generation sequencing technologies in a CLIA-environment, is among only a handful of commercial labs in the U.S. currently offering testing for inherited cancer and full genome testing for targeted genes, such as GeneDx Cardiology.

GeneDx‘s cancer diagnostic menu includes:

  • Comprehensive Cancer Panel (35 genes)
  • Breast/Ovarian Cancer Panel (26 genes)
  • Breast Cancer High Risk Panel (6 genes)
  • BRCA1/2 Ashkenazi Founder Mutation Panel BRCA1/2 Sequencing and Del/Dup Analysis Colorectal Cancer Panel (17 Genes)
  • Lynch/Colorectal High Risk Panel (7 genes)
  • Pancreatic Cancer Panel (16 Genes)
  • Endometrial Cancer Panel (11 Genes)

Their cardiac disease specialty includes:

  • Comprehensive Cardiomyopathy Panel (seq & del/dup 76 genes)
  • Comprehensive Arrhythmia Panel (seq & del/dup 30 genes)
  • Pulmonary Arterial Hypertension Panel (seq & del/dup 5 genes)
  • Hereditary Hemorrhagic Telangiectasia (seq & del/dup 4 genes)
  • Long QT Syndrome (seq & del/dup 12 genes)

Other NGS panels include:

  • The Marfan/TAAD Next-Generation Sequencing Panel includes 16 genes (ACTA2, CBS, COL3A1, COL5A1, COL5A2, FBN1, FBN2, FLNA, MED12, MYH11, SKI, SLC2A10, SMAD3, TGFB2, TGFBR1, TGFBR2)
  • The Comprehensive Maturity-Onset Diabetes of the Young (MODY) Panel analyzes 5 genes (GCK, HNF1A, HNF1B, HNF4A, PDX1) associated with MODY
  • The Comprehensive Severe Combined Immunodeficiency (SCID) Panel analyzes 18 genes associated with SCID disorders
  • The Comprehensive Mitochondrial Nuclear Gene Panel analyzes 101 nuclear genes for clinical molecular diagnosis of mitochondrial disorders

The GeneDx Prenatal Targeted Array is a combined CGH and SNP array for detecting copy number changes and uniparental disomy (UPD), and GeneDx’s XomeDx is designed to identify rare inherited diseases.

The LiDia diagnostic platform—formerly the Genalysis system—by DNAe, is a sample-to-result genomic analysis platform based on semiconductor sequencing, capable of performing both parallel individual assays and more complex NGS. It is an integrated instrument and consumable cartridge that will deliver results directly from blood in 2-3 hours, using a simple electric signal from hydrogen ions released in DNA amplification, detected by a silicon transistor. In September 2017, the company revealed a pipeline of upcoming rapid diagnostic tests for the platform; the first test that will be available is the LiDia bloodstream infection (BSI) test for use in sepsis diagnosis and management, testing for bacterial and fungal pathogens as well as antimicrobial resistance. Applications anticipated for release further in future include antibiotic-resistant infections, flu, and cancer.

Through a non-exclusive license to Thermo Fisher Scientific, DNAe’s semiconductor sequencing technology is the core of the Ion Torrent NGS systems, proving its performance at the laboratory scale.  For POC, DNAe’s semiconductor sequencing is amenable to nano-scale consumer applications, delivering sequence information from a device the size of a USB stick.

FoundationOne, the first clinical product from Foundation Medicine, is a fully informative genomic profile for solid tumors used to identify the molecular alterations in a patient's tumor using next-generation sequencing in routine cancer specimens. The FoundationOne test can detect genomic alterations in more than 230 genes that are thought to be most relevant to the growth and spread of cancer. FoundationOne interrogates all genes somatically altered in human cancers that are validated targets for therapy or unambiguous drivers of oncogenesis based on current knowledge.

FoundationOne uses routine, formalin-fixed, paraffin-embedded tumor samples. Test results are provided in a report that matches detected patient’s genomic alterations with potential treatment options and clinical trials. To date, Foundation Medicine has performed the test on lung, colon, breast, endometrial, GI, salivary, liver, and kidney cancers, on soft tissue sarcomas, esophageal and gastric neuroendocrine tumors, and with basal cell carcinoma.

Illumina’s new flagship NovaSeq 6000 launched in March 2017. It can be equipped with two of four types of flow cells: S1, S2, S3, and S4. Dependent upon which is in use, Novaseq is capable of 1.6-20 billion reads and throughputs of 480-6000 Gb per flow cell, with a runtime of 44 hours, capable of producing three to 48 human genomes per run. In addition to whole genome, exome, and transcriptome sequencing, applications for the platform include target gene, methylation, and de novo sequencing, gene expression profiling, and metagenomics, among others. Illumina president Francis deSouza expressed a multi-year aim for NovaSeq to replace the 1,900 HiSeq instrument install base, explaining that NovaSeq provides a 20% reduction per Gb and three times the throughput over the former high-end HiSeq X, with even greater efficiency over the rest of the line; unlike HiSeq X, there will be no minimum order necessary for NovaSeq. Illumina also states that the scalable sequencing architecture used in NovaSeq will one day lead to future systems that can generate a $100 genome.

As the company’s name implies, Oxford Nanopore Technologies’ instruments operate on tech based on nanopore sensing, in which nanopores are set in an electrically resistant membranes and ion currents are passed through them. As an analyte passes through a pore or near its aperture, a characteristic disruption in the current is created. This can be measured, making it possible to identify the analyte. For DNA sequencing, the sequencing systems can be used to distinguish between the four standard DNA bases G, A, T and C and modified bases. With the GridION system, five nanopores can be simultaneously measured, and data can be sensed, processed and analyzed in real time, generating 100 gigabases of data in 48 hours. The PromethION is a tablet-sized benchtop instrument designed to run a few samples on a very large number of nanopores or, instead, to run multiple samples in parallel with fewer (~thousands) of nanopore-sensing channels each.

The MinION device is a small, hand-held device that is compatible with consumable flow cells containing the proprietary sensor chip, Application-Specific Integrated Circuit (ASIC) and nanopores that are needed to perform a complete single-molecule sensing experiment. When plugged directly into a laptop or desktop computer through a USB port, the MinION is operated as a self-contained device that can deliver real-time experimental data.

A fourth instrument, the tiny SmidgION, is currently under development. This device offers genome sequencing using the same nanopore sensing technology as MinION and PromethION, but is small enough to be attached directly to an iPhone via Lightning port. Also recently announced is the development of Flongle, a flow cell dongle adapter for MinION and GridION that enables real-time nucleic acid sequencing on smaller, single-use flow cells. No ETA is available for these two products.

QIAGEN relaunched its comprehensive next-generation sequencing platform, the GeneReader system, for the US market in November 2016; the system’s development stemmed from the company’s 2012 acquisition of Intelligent Bio-Systems. With new and improved sequencing chemistry for the system, QIAGEN was able to report 100% concurrence in an internal study testing the system against two of the company’s own PCR assays and with a third party’s NGS sequencer. The GeneReader performs NGS applications by integrating highly-parallel fluorescence-based sequencing chemistry with detection of the corresponding fluorescent signal templates that have been clonally amplified using the GeneRead QIAcube.

GeneReader automates sequencing primer hybridization, reagent prep, experiment setup, and flow cell prep, loading and run start. The device’s workflow also includes a post-run maintenance wash. Amplified DNA is immobilized via direct bead-slide interaction and exposed to a sequencing primer, producing a high-density array on a flow cell. The array is then exposed to a number of reagents that contain DNA bases and include a removable fluorescent dye and end cap, which attach themselves to the end of the expanding DNA strand according to the base on the complementary strand. A high-resolution camera scans the array and measures the fluorescent output of the dye colors in each array position, indicating which nucleotide base was incorporated onto the DNA fragment. Cleavage chemistry then breaks the fluorescent dye and the end cap off of the fragment to allow further augmentation with additional bases, and the cycle begins anew.

Roche acquired Genia Technologies in June 2014 to develop NGS technology with improvements to cost and sequencing speed. Genia’s platform replaces complex, expensive optical sensors with integrated circuits to detect nucleotide base tags during single molecule DNA sequencing, and  combines them with a nanopore array. Software on the chip controls the insertion of nanopores into lipid bilayers and the active control of individual sensors on the array. Cleaved tags from sequencing provide specific signatures corresponding to each of the four nucleotide bases. Each bioengineered nanopores can sequence a single piece of DNA, with thousands performing massively parallel sequencing. The first prototypes of the Genia sequencer feature an expanded chip with as many as 128,000 sensors. Genia aims to introduce a sequencer capable of sequencing an entire patient genome at a cost of $100. The platform, which is still under development, will also perform sequencing without standard library preparation and provide genome sequencing results within hours.>

Kalorama Information will be attending American Association for Clinical Chemistry's (AACC) annual meeting in Chicago to speak with in vitro diagnostic and related vendor companies. To set up an appointment with Kalorama’s team, contact our Publisher,Bruce Carlson.

Image attribution: Next Generation Sequencingby Jenny Cham, on  flickr (Creative Commons license)