The genomics industry has come a long way since the human genome project, which relied so heavily on Sanger sequencing with capillary electrophoresis to unlock sequences of base pairs in human DNA. There has been an increasing focus on efficiencies and driving down sequencing costs, resulting in the achievement of the '$1000 genome'. Now that sequencing costs have declined so significantly, the focus of many technology vendors has moved onto sample preparation and informatics: get a better quality sample in, quicker, and get an easier to understand answer out. This was evidenced at AGBT 2016 in Orlando Florida, where there was a strong focus on the workflow: 'sample in, insight out'.
The SIRVs are a set of 69 artificial transcript variants which mimic 7 human model genes. They are complemented by additional isoforms to reflect diverse variations of RNAs in cells. The accuracy of mapping, isoform assembly and quantification can be assessed, making isoform-quantification based experiments comparable.
"The performance of RNA-Seq cannot be properly assessed without known transcript spike-in controls of representative complexity. To address this gap, Lexogen has conceived Spike-In RNA Variants (SIRVs) for the quantification of mRNA isoforms in Next Generation Sequencing (NGS)."
Jungsoo Park, Head of Marketing and Sales
With budgets and timelines tighter than ever, there is a growing need for more efficient workflows in laboratories. At the same time, there is increasing interest in analyzing copy number variation (CNV) from NGS data. Agilent OneSeq Target Enrichment offers a simple, streamlined, cost-effective solution for detecting single gene mutations, CNVs, and aneuploidies in one assay.
Hot Topics from the Conference Floor:
Keeping Pace with the Needs of the Genomics Market:
AGBT is a place for unveiling and previewing the future in genomics technology, with many companies choosing to give a sneak peek under the hood of what they are working on. Illumina went so far as to give a preview of its latest project - Firefly - which will look to combine sample prep and sequencing in one small footprint semiconductor-based instrument. Not planned to launch until 2017 at the earliest, they are mentioning it now as the instrument will act as a platform on which third party apps can be deployed and would therefore be very difficult to keep secret.
Fluidigm has a similar model with its C1 instrument that also features a community area (called the C1 Open App Program), where third party apps can be deployed. This has two functions: to provide a platform where customized workflows for chip and software technology can be shared as soon as they become available, ensuring the hardware does not become obsolete; and secondly to enable better collaboration of apps that are developed by third parties (including users) and deployed on the C1.
Both announcements play into two major themes of the conference: future proofing technology and the challenge vendors face keeping up with the rapid advances in genomics.
Collaborations and Partnerships:
The challenge of keeping pace with the technological needs of users makes it increasingly difficult for vendors to remain technology specialists across the product spectrum, a key reason why AGBT is a platform for announcing multiple partnerships and collaborations.
Agilent Technologies and 10X Genomics announced they are combining expertise in sample prep and target enrichment with long range sequencing products, to enable customers to ‘look beyond the exome’. This means users will be able to look at not just structural variations, mutations and previously unmappable loci, but also translocations that result in human disease. The companies will be combining complementary products - 10X Genomics' Chromium platform and Agilent's SureSelect - into a workflow to produce a 'premium exome'.
10X Genomics announced a number of collaborations at AGBT, which included a partnership with Illumina to enable long read applications and QIAGEN for co-marketing and co-development of sequencing and single cell analysis workflows as well as a variety of informatics solutions. Other partnerships were highlighted, with Invivoscribe announcing a collaboration to develop immuno-oncology tests with Thermo Fisher Scientific; Children’s Hospital Los Angeles (CHLA) and Thermo Fisher, who agreed to develop a next-generation sequencing (NGS)-based panel designed specifically for pediatric cancer research; and between Illumina and Bio-Rad.
One of the main focuses of this year's AGBT was on the use of genomics in research and the clinic. Within this, there were four big application areas that really stood out:
- Clinical diagnostics, especially with the advent of liquid biopsy
- Single cell analysis
- The drive to 'go beyond the exome'
- The ability to sequence increasingly longer nucleic acid fragments.
Traditional biopsy methods are uncomfortable and often associated with negative side effects. Liquid biopsy, in contrast, is non invasive and carries little known side effects. This enables it to be used regularly to monitor, guide treatment and provide valuable genomic information about a disease. All factors which are giving rise to its increasing use, especially within personalized medicine.
Liquid biopsy technologies are now being incorporated into the sequencing workflow for maximum efficiency. To this end, Cynvenio announced an extension of its partnership with Thermo Fisher, which will sell Cynvenio's Concordia™ cell-free DNA (cfDNA) purification sample preparation upgrade for its multi-template analysis LiquidBiopsy® platform. The upgrade package enables users to purify DNA from whole blood using Thermo Fisher's Applied Biosystems MagMAX cell-free DNA isolation kit. When coupled with Thermo Fisher’s Ion Torrent™ next-generation sequencing (NGS) systems, Cynvenio’s workflow provides a comprehensive molecular characterization of tumor cell DNA for cancer research samples from a standard blood draw.
QIAGEN also focused on liquid biopsy, highlighting its portfolio of solutions to enable the development of emerging liquid biopsy technologies in clinical research and diagnostics. It claims to be the only company addressing all three liquid biopsy approaches: free-circulating nucleic acids, circulating tumor cells, and exosomes.
Single Cell Analysis:
Single cell analysis enables cell and molecular biologists to understand the difference in gene expression profiles between individual cells. As opposed to the previous practice of taking averages of entire cell populations, single cell analysis accounts for the critical changes that occur in individual cells. The technique also enables the study of previously undetected subpopulations within complex samples.
To help with this understanding of complex cell dynamics through evaluation of individual cells, WaferGen Biosystems developed its ICELL8 Single-Cell system to enable users to isolate thousands of single cells, select specific cells of interest and analyze them regardless of their type and size. This provides control of cell selection as well as providing insight into the cells via the ability to evaluate up to 8 samples on a single chip and discover unique cell populations.
Similarly, Fluidigm’s Polaris is the first system to integrate cell and molecular biology at the single-cell level. It enables customers to select their target cells by phenotype, select dose and prepare cells for analysis. Polaris allows researchers to keep the cells alive and interrogate them for up to 24 hours and then measure each cell’s acute response transcriptionally via next-generation sequencing.
BD’s Molecular Indexing technology, when combined with its fluorescence activated cell sorting (BD FACS) technology, was demonstrated as a cost-effective workflow for single-cell mRNA sequencing. The combination of the two technologies results in a powerful tool for both enrichment of input for a more homogenous sample and linking cell phenotype with molecular gene expression profiles.
Longer molecule fragments:
Researchers increasingly want to sequence longer molecule fragments for downstream applications such as long read NGS and DNA restriction analysis. These longer fragments enable researchers to look at the whole sequence in context making the process less time and labor intensive when compared to the current methods for analyzing quality of large fragment DNA, such as agarose gel electrophoresis and pulsed-field gel electrophoresis.
To address some of these challenges associated with existing techniques, Advanced Analytical launched a large fragment analysis kit for its Fragment Analyzer Automated CE System. The DNF-464 High Sensitivity Large Fragment 50 Kb Kit, provides improved DNA sample analysis in shorter run times (about 55 minutes of total electrophoresis time) enabling accurate assessment of large fragment Pac Bio libraries. Also, shown for the first time was the FEMTO Pulse, which the company claims will revolutionise nucleic acid detection by reducing detection time from approximately 18 hours to around an hour and increasing detection sensitivity to the single femtogram levels. Advanced Analytical developed the underlying Pulse Field technology to work with its award winning parallel capillary array design to analyse nucleic acid fragments and smears.
Now that sequencing technology is no longer in its infancy, we have seen vendors shift their focus from sequencers to sample prep and informatics, combining them in an automated and intelligent workflow. Here are some of the highlights from the vendor suites:
Sample In: Answer Out – The Rise of the Workflow:
In addition to the workflow highlighted by Agilent and 10X Genomics, Roche also unveiled its new end to end sequencing solution that is currently in development. The company highlighted how their 2014 acquisition of Bina Technologies has added advanced informatics capabilities, including the ability to process ’big’ NGS data in one centralized management system.
QIAGEN highlighted a complete ‘sample to insight’ sequencing workflow that features its GeneReader NGS System. A fully automated workflow, it includes sample prep; next gen sequencing with the new GeneReader; and finally analysis and interpretation of the data with the QIAGEN Clinical insight (QCI) platform.
The automation of sample preparation, specifically nucleic acid extraction, library preparation and target enrichment were key themes from most of the vendors. It is all about getting better quality samples to work with, and making their preparation as efficient and easy as possible. As noted by Heidi Kijenski, Director of Product Marketing Diagnostics and Genomics Group, Agilent, ‘sample quality has a direct impact on the fidelity of sequencing data’.
The company offers a new target enrichment panel for combined detection of LOH (loss of heterozygosity), targeted mutations and genome-wide copy number changes. Called OneSeq, the panel enables the cost-effective and efficient discovery of new genes associated with syndromes and allows clinical researchers to investigate the role of variations in copy numbers and mutations in some genetic disorders. This is part of a range of catalog and custom target enrichment products that includes SureSelect (a hybridization-based technology), ClearSeq cancer research panels for detection of somatic variants in solid tumors and hematological cancers; and HaloPlexHS, a PCR method that uniquely tags each DNA library with molecular bar codes to track molecules through the library preparation process.
In addition, it highlighted the Agilent 4200 TapeStation. Launched in October 2015, the TapeStation enables end to end automated sample QC in an NGS workflow (for research use only). The result, it is claimed, is both higher throughput and quality improvements of sample.
Fluidigm’s Juno was highlighted as a solution for targeted DNA next-generation sequencing library preparation. The company explained that preparing the sample can be more time consuming and expensive than running the sequence itself. Juno has been designed to automate library preparation for difficult to use samples and can produce hundreds of targeted DNA sequencing sample libraries daily, with each sample enriched for up to 2,400 specific amplicons covering user-defined genomic loci. Designed mainly for clinical applications, Juno will enable researchers and eventually clinicians to get to a result faster and more affordably that previously possible. Covaris unveiled its ME220 Focused-ultrasonicator to provide batch processing at the benchtop. It employs AFA technology (Adaptive Focused Acoustics) to provide non-contact isothermal sample preparation.
New England Biolabs highlighted its recently launched NEBNext FFPE repair mix. The methods used for fixation and storage of FFPE samples are common practice but can damage the quality of the resulting nucleic acids. The Repair Mix is a formulation of enzymes that repair DNA. For non-FFPE samples, it is hard to extract and keep longer molecule fragments without damaging them, and the Repair Mix formula can also be used to treat and repair long DNA molecules before sequencing.
The Ultra II kit for DNA library preparation from New England Biolabs got its first showing at AGBT. Designed for preparing low input, poor quality or ‘difficult’ samples for sequencing, as well as high quality samples, the Ultra II can produce several-fold higher library yields with as little as 500 picograms of human genomic input DNA.
PerkinElmer showcased its Chemagic 360 for compact, high volume nucleic acid isolation. The Chemagic 360 is more sensitive than traditional filter-based extraction products. It can uniquely process sample volumes from 10 μl - 10 ml on one single instrument, bringing potential cost savings of up to 50% if both high and low volume samples are processed. The company also highlighted its Janus Automated Liquid Handling series that can be combined with the Chemagic 360 to provide a fully automated, high throughput, nucleic acid extraction solution.
BD also highlighted its BD FACSeq Cell Sorter with BD Precise Assays for high precision single cell gene expression analysis (launched in October 2015). This workflow for gene expression studies of thousands of single cells provides high quality data as well as a solution to isolate 96 single cells in less than 30 seconds and an NGS-ready library in 5 hours. The company also unveiled the BD CLiC System which it claims is the first high-throughput, fully integrated, automated bench-top library preparation workflow for NGS targeted enrichment and whole genome library preparation.
Advanced Analytical showcased its recently launched Fragment Analyser INFINITY Capillary Electrophoresis system. This new instrument automates the sizing, quality and quantity analysis of nucleic acids, and can interface with a robotic arm for continuous operation.
Thermo Fisher Scientific highlighted the Ion Chef System that is all about convenience and automation. The system is designed to automate Ion AmpliSeq library preparation, template generation and chip loading, to produce data in around 45 minutes of hands-on time.
Lexogen highlighted its Spike-in RNA Variants Mixes (SIRVs) that were originally launched in September 2015. Spike-in controls are essential in RNA-Seq experiments to assess workflow and platform properties. However, asJungsoo Park, Head of Marketing and Sales atLexogen explains, “The performance of RNA-Seq cannot be properly assessed without known transcript spike-in controls, of representative complexity. To address this gap, we conceived Spike-In RNA Variants (SIRVs) for the quantification of mRNA isoforms in Next Generation Sequencing (NGS).” The SIRVs are a set of 69 artificial transcript variants which mimic 7 human model genes. They are complemented by additional isoforms to reflect diverse variations of RNAs in cells. The accuracy of mapping, isoform assembly and quantification can be assessed, making isoform-quantification based experiments comparable.
NanoString Technologies took the opportunity to demonstrate proof of concept for a new chemistry for single molecule sequencing that is being called Hyb & Seq. The resulting workflow does not require enzymes, amplifications or libraries, opening up the possibility of a sample-to-answer sequencing instrument appropriate for clinical use.
Roche previewed a new sequencer that is based on the PacBio SMRT technology. The aim is to launch the product in H2 2016. Roche will take its experience in the clinical market and combine it with the SMRT sequencing capabilities to produce a workflow that features dedicated kits designed specifically for use in a clinical research environment.
Thermo Fisher Scientific showcased its new Ion S5 XL designed for simplified NGS in clinical and translational research. The Ion S5 XL (and its lower throughput sister Ion S5) are designed to provide a simple DNA-to data workflow for targeted sequencing. The company demonstrated how, when combined with the Ion Chef and the Cynvenio Liquid Biopsy platform, the result is a simplified sequencing workflow that is easy to use and cost effective.
10X Genomics launched the Chromium System, which is an upgrade for existing short read sequencers. Based on the GemCode technology, the system provides long range information, megabase-scale phasing, uniform coverage and structural variant detection while integrating single cell sequencing into the same system.
Illumina showcased the recently launched MiniSeq System. To address the challenge of making the sequencing easier and more cost effective, the MiniSeq System is designed with push-button operation and, at a price point of $49,500 USD, it should bring it within range of most labs.
With NGS instruments producing huge amounts of data, the informatics needed to manage and interpret this has historically caused a bottleneck in the sequencing workflow.
The strong focus on informatics on show at AGBT this year, demonstrated the commitment of vendors to address this challenge. This focus was on both new informatics solutions as well as cloud-based services, with Thermo Fisher and Roche having their own Amazon-based cloud solution.
In mid 2015 Agilent acquired Cartagenia, a software provider that enables clinical analysis of genomics data. The company highlighted at AGBT that the integration of Cartagenia into the NGS workflow will provide customers in a clinical setting with the ability to visualize, assess and report clinical genetics data in the context of patient information.
QIAGEN put a spotlight on the bioinformatics aspect of its workflow, which is designed to support customers to perform quality checks and secondary analysis. Launched in December 2015, the Clinical Decision Support Tool provides a clinical lab director with the ability to interpret variants and pair them with the right health options for precision medicine.
In summary, the genomics technology market is both innovative and fast paced. There are exciting new technologies both available for purchase now and in the development pipeline. It is evident that there will continue to be more consolidation of vendors both through partnerships and full acquisitions, resulting in the holy grail of integrated workflows that feature instruments from a range of specialists. These workflows will automate the sequencing process and really enable ‘sample in: answer out’ capabilities at a low price point.