Advances in Genomic Analysis Technologies: An Industry Guide

Introduction:

The structure of DNA was discovered over 60 years ago, yet many mysteries remain on how our genome functions. In order to understand what the genome means we need to know, not just its sequence, but how this relates to phenotype. Our genome is over three billion bases long, meaning that genome sequencing produces vast quantities of data that must be deciphered and, historically, this has been a slow process. But, advances in genetic sequencing technology mean that we stand on the edge of a revolution that will see genetic sequencing data be quicker to obtain, easier to decipher and increasingly informing medical diagnosis and treatment.

Not only have recent years seen major advances in genetic sequencing but they have also witnessed the emergence of the genetic-editing technology CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). The arrival of CRISPR has given scientists their most versatile genome-editing tool yet, allowing us to edit the genome at specific locations quickly, cheaply and accurately. As a result, the realms of genetic engineering that have been hypothesized and anticipated for the future have suddenly become today’s reality.

Genomics – the study of genomes – is a branch of science that is finding its own in the 21st century. Although our understanding of genetics has informed a multitude of medical discoveries and treatment, in the coming years this role is going to explode to become a central pillar of healthcare. The catalyst that has fueled this is next-generation-sequencing and the company that has been at the forefront of this revolution is Illumina.

This year’s Pittcon, taking place in Chicago from 5-9 March, 2017, will be hosting The Twenty-Eighth James L Waters Symposium on Genomic Analysis Technologies. This prestigious annual symposium was founded to explore the origin, development, implementation, and commercialization of scientific instrumentation of established and major significance. And in 2017, it will highlight Illumina’s technologies.

It will feature speakers from Illumina itself, including co-founder David Walt and long-time CEO and now executive chairman, Jay Flatley, as well as leading genetic analysis companies that are using Illumina’s instruments to facilitate consumer and healthcare genetic testing.

Also at the Pittcon exposition will be a number of leading companies delivering genetic sequencing solutions. This year, the list includes Thermo Fisher Scientific, Eppendorf, Beckman Coulter, Vitl Life Science Solutions, Panasonic Healthcare, and Malvern Instruments.

We look forward to seeing you in Chicago in March 2017!


Chapter 1 – Next Generation Sequencing

On 26th June 2000, Bill Clinton, then President of the United States, announced to a press conference at the White House that the first “rough draft” of the complete human genome had been sequenced as part of the Human Genome Project. But while this was a landmark achievement in genomics, in the intervening years such remarkable progress has been made in the field of genetic sequencing, that it essentially belongs to another era.

The entire Human Genome Project took over 10 years to complete at a cost of $3 billion. Now the technology we have can process 20,000x as much data, and a person can have their entire genome sequenced for just a thousand dollars.

Genetic information is now poised to play a central role in our healthcare, with decisions made in doctors’ offices day-to-day expected to increasingly be informed by sequencing results. Direct-to-consumer genetic testing is also on the rise, and soon we will see prenatal and cancer diagnoses routinely made via blood tests rather than invasive procedures. Furthermore, the vast wealth of data that can now be rapidly acquired is going to inform our genetic understanding of health and disease at an unprecedented level.

Since the first publication of the human genome sequence, whole genetic technologies have come and gone. But one has emerged to dominate over the rest: Illumina sequencing. The company is now thought to hold around 90% of the genetic sequencing market share.

Pittcon 2017 will be hosting The Twenty-Eighth James L Waters Symposium on Genomic Analysis Technologies. This year, the symposium will focus on Illumina’s history and technologies, while looking to the future applications of this science.

The symposium will be fortunate to hear from both Dr David Walt, one of Illumina’s co-founders, who will describe the journey from academic discovery, to startup, to market-leading company. The symposium will also be joined by Jay Flatley, executive chairman of the board of directors, who served as CEO for 17 years and oversaw the company’s acquisition of its flagship next generation sequencing technology Solexa, as well as its rise to dominance in the market.

Although the principles of Illumina’s sequencing by synthesis, or SBS, technology have remained the same, since acquiring Solexa 10 years ago, the company has continued to invest in its innovation to keep pushing the limits of what it can do. The most recent generation of Illumina SBS instruments can generate 1.8 terabases of data per run compared with one gigabyte just a decade ago.

There will also be even more genetic sequencing technology companies at the Pittcon exposition, including Thermo Fisher Scientific, Beckman Coulter and Eppendorf.

Thermo Fisher Scientific provides next-generation sequencing technology through its Ion Torrent semiconductor sequencing products. In addition to hardware, Thermo also offers a complete next-generation sequencing data analysis package in the form of the Ion Reporter software and server. Applications for the technology include targeted DNA, transcriptome, targeted RNA, and exome sequencing.

We will also be joined at Pittcon 2017 by Beckman Coulter. Beckman’s SPRIworks HT provides high-throughput sample preparation for Illumina next-generation sequencing platforms. This technology helps to facilitate the increased throughput of next-generation sequencing by reducing the labor intensity and hands-on time required for library preparation. This is achieved through an automated workflow using Solid Phase Reversible Immobilization (SPRI) paramagnetic bead based technology. It is able to process up to 96 samples in as little as three hours.

Eppendorf will also be presenting at the Pittcon 2017 exposition. The automated workstations from Eppendorf, such as the epMotion 5075t / 5075m, can be incorporated into an Illumina sequencing workflow. The automated pipetting system comes with built-in ThermoMixer comes with a range of pre-programmed and validated next-generation sequencing library preparation workflows and can process up to 96 samples in a day.

References


Chapter 2 – DNA Screening

The advent of low-cost next-generation-sequencing is opening new doors in DNA screening, creating the possibility of non-invasive cancer detection, prenatal diagnosis and genetic carrier testing via blood samples.

At the James L Waters Symposium on Genomic Analysis Technologies, we will hear from some of the leading players in this burgeoning industry. This includes GRAIL, an Illumina spinout that is developing blood screening tests for cancer in asymptomatic people, sometimes referred to as ‘liquid biopsies’.

Also presenting at the symposium will be LabCorp, who focus on development of non-invasive prenatal testing, or NIPT. These tests work by detecting DNA shed from the placenta and could help lower the use of amniocentesis for prenatal diagnosis – an invasive procedure that carries a substantial risk of miscarriage.

Genetic screening is also increasingly being chosen by consumers to make decisions about their health. One of the frontrunners in this field is Counsyl, who will also be at the James L Waters Symposium. They are trying to make genetic screening cheaper and more accessible to all. The company offer DNA screening and genetic counseling for inherited conditions and cancer risk genes, and prenatal screening, with some of their services available online.

At the James L Waters Symposium, Alex Aravanis who heads up research and development at GRAIL will speak about the challenges of developing a safe and effective cancer screening test. He will also explain how advances in next-generation-sequencing are enabling the detection of circulating nucleic acids shed from tumor cells in blood samples. The company’s aim is to produce ways of detecting cancers much earlier, to increase the likelihood that they can be effectively treated and enhance survival.

Daniel Grosu from LabCorp will also be speaking on how a similar approach is leading to the development of non-invasive prenatal testing, or NIPT. The company launched their own InformaSeq test in 2014 and in 2016 completed acquisition of Sequenom, who were the first to offer prenatal non-invasive commercial sequencing for chromosomal aneuploidy.

There will also be a presentation from Dr Jim Goldberg, Chief Medical Officer at Counsyl. The company offers three main products: a genetic carrier screen that covers more than 100 inherited disorders, a non-invasive pregnancy screening test, and a hereditary cancer screen that looks at 36 cancer-related genes, including for breast, ovarian and colon cancer.

References


Chapter 3 – CRISPR

During the last five years, one major development in gene editing technology has seized the world of biology with excitement. CRISPR/Cas9 has made genome editing more specific and easier than ever before, giving us an unprecedented ability to manipulate DNA.

The arrival of CRISPR has opened up myriad possibilities, including the potential to conduct experiments that were previously impossible. It can give us new insight into disease by allowing scientists to knock out or modulate specific genes and study their function, it can be used in drug development, such as the creation of new antimicrobials, and work has already begun to apply the technique clinically to cells of the human body. Outside of biomedical applications, CRISPR will also find uses in agriculture and livestock breeding, and in environmental settings such as control of disease-vector insects using gene drives.

At this year’s Pittcon, you have the opportunity to meet the companies who are delivering the tools and technology to put CRISPR to use in your own lab. These include ThermoFisher Scientific, Eppendorf, Beckman Coulter, Vitl Life Science Solutions, Panasonic Healthcare, and Malvern Instruments.

What is CRISPR?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat. These are clusters of DNA repeats that were first identified in bacterial genomes in the 1980s. Research revealed that they are part of the bacterial adaptive immune system. When a bacterium is infected by a virus, it can incorporate short sequences of DNA from the genome of the invading virus into its own genome. As such, they serve as a memory of past viral invaders. These sequences are stored as “spacers” in between CRISPR sequences. Then, if the same virus attacks the bacterium at a later date, it can recognize and cut any section of the invaders genome matching the spacer sequence. To do this, the transcribed CRISPR sequences, composed of single-stranded RNA, act as guides for the enzyme Cas9, which has the ability to cut through DNA. As a result, it is able to disrupt and disable the viral genome at very specific locations.

The CRISPR/Cas9 gene-editing system hijacks this ancient process by using guide RNAs, which are short synthetic stretches of RNA, to guide the Cas9 enzyme to a target location in the genome. This has created a powerful tool that allows specific genomic sequences to be inserted, deleted or replaced.

Facilitating CRISPR Experiments

At Pittcon 2017, we will be joined by some of the world’s leading biotechnology companies that provide solutions for each stage of the CRISPR protocol. These include: Eppendorf, offering a range of equipment for multiple stages of CRISPR preparation including thermo-mixing devices, centrifuges, thermocyclers and CO2 incubators; Thermo Fisher Scientific offering ready-to-use CRISPR/Cas9 products including vectors, Cas9 mRNA and protein and CRISPR libraries; as well as of course Illumina, offering next-generation sequencing products that are ideal for library verification and validating genome edit success.

The CRISPR process

Vector Generation

Pittcon exhibitor Thermo Fisher Scientific offer the GeneArt CRISPR Nuclease Vector Kit, which is a ready-to-use, all-in-one system that makes it easy to encode a guide RNA using a vector that also expresses the Cas9 enzyme.

Transformation and Cultivation

Centrifugation is an ideal method to concentrate lentiviral vectors for use in CRISPR experiments. Pittcon exhibitor Beckman Coulter offer a range of centrifuge devices for this purpose including their Optima X series ultracentrifuges which range from comprehensive floor models to the tabletop Optima MAX-XP.

Vitl Life Science Solutions, will also be demonstrating the Co-Mix laboratory mixer at this year’s Pittcon exhibition. The device allows you to mix plates and vortex them at the same time, thanks to its unique independent vortex pad, and can be used in a variety of genetics experiments and PCR.

Preparation and Purification

The Vitl Life Sciences Ther-Mix is a fully programmable heater laboratory mixer with numerous applications including in DNA isolation, plasmid isolation, and sample prep for PCR. It allows you to combine multiple heating and mixing steps in one device to reduce variability and contamination risk.

Transfection and Cultivation

One key application of CRISPR is for the generation of genetically modified animals, which can provide insights into gene function, disease mechanisms and assist drug development. The Eppendorf TransferMan 4r is a multifunctional manipulator that can be used for the injection of CRISPR/Cas9 components into the cytoplasm or pronucleus of oocytes or embryos.

In an ideal workstation setup, it can be used alongside the Eppendorf PiezoXpert, which uses piezo impulses to ensure gentle and effective penetration of the cell membrane. Piezo-assisted micromanipulation improves results of a variety of applications, including transfer of embryonic or induced pluripotent stem cells into blastocysts, mouse ICSI (intracytoplasmic sperm injection), and enucleation/nuclear transfer.

Pittcon 2017 will also be attended by Panasonic, that offer a range of CO2 incubators that can provide accurate and tightly controlled conditions for cell incubation.

Harvesting and Preparation

When the time comes to harvest cells during CRISPR experiments ready for analysis, a complete set of tools and technologies are on offer from the leading manufacturers. For example, Eppendorf provide centrifuges, tubes and plates, and a range of purity grade consumables to safeguard against contamination by any biological substance. The Eppendorf ThermoMixer C can also heat, cool, and mix almost any lab vessel.

Analysis and Detection

A photometer is an ideal tool for verifying RNA and DNA concentration during CRISPR experiments. Eppendorf, an exhibitor at Pittcon 2017, offers a well-established line of photometers, including the BioPhotometer D30 and the Biospectrometer Basic, for rapid and accurate nucleic acid photometry.

And when using PCR to amplify mutated regions and target sites, the Eppendorf Dual Block Thermocycler Mastercycler nexus X2 can help reduce variability and ensure consistent results. The device is perfect for busy labs by providing two separate thermoblocks, allowing you to run two separate PCR experiments simultaneously, but completely independently.

References


Chapter 4 – CRISPR Applications

The ability to easily and efficiently modify genetic sequences within cells holds enormous potential across basic science, biotechnology and medicine. Via the application of CRISPR, life sciences research can generate a better understanding of how genes and regulatory elements function and reveal how the genome is organized within the cell. In the field of biotechnology, genetic engineering could be used to create infection-resistant crops. And within medicine, genome engineering could lead to a whole new generation of therapies, and improved models of disease. There is also the possibility to apply the technology directly as gene therapy to correct harmful mutations.

Previously studying the physical organization of the genome required denaturation, making it virtually impossible to study dynamic processes in living cells, and fluorescent tagging of DNA in live cells has mostly been restricted to specific chromosomal regions, such as the centromere or telomere. CRISPR has created an opportunity to study DNA within living cells through the adaptation of genome-editing tools. Due to the relationship between genome structure and function, such methods could therefore offer invaluable insight into the genome’s workings.

At Pittcon, we will be joined by some of the leading manufacturers who are providing the tools needed to put CRISPR into action in the lab. For example, Eppendorf will be presenting the micromanipulation system, with PiezoXpert, which can help successful pronuclear injection.

Piezo–assisted injection methods can significantly increase productivity and enhance yields of cell micromanipulation experiments. The system works with piezo impulses which are directly transferred to the capillary to generate perforation of the cell membrane. It can be applied to a variety of applications including enucleation/nuclear transfer, as well as transfer of embryonic or induced pluripotent stem cells into blastocysts and morulae, and injection into mammalian oocytes and zygotes.

Pittcon 2017 exhibitor, Thermo Fisher Scientific, provides a complete toolset for every step of the genome editing workflow, whether for generating animal disease models, stem cell engineering or creating transgenic plants. Thermo Fisher produce an all-in-one CRISPR genome editing solution that includes an online CRISPR search and design tool, four formats of CRISPR/Cas9 (Cas9 protein, Cas9 mRNA, all-in-one expression vector, and CRISPR Libraries) as well as cell culture reagents, delivery method, and analysis tools. They provide a demonstrated protocol for the use of their CRISPR/Cas9 products in human induced pluripotent stem cells, one of the major applications of the genome-editing technology in research. And the company have shown that their GeneArt CRISPR-Cas9 System is able to perform edits in both mouse embryonic stem cells and human induced pluripotent cells with over 50% efficiency.

Thermo Fisher Scientific have also demonstrated the use of genome-editing tools in the creation of animal models. A team tested the efficiency of both Invitrogen GeneArt Cas9 recombinant protein, or Cas9 mRNA and in vitro-transcribed short guide RNA (sgRNA) in the generation of genetically engineered mouse models by carrying out pronuclear injections and culture of zygotes at the blastocyst stage. The team say the approach is suitable for multiplexing and they have had success in testing between six and eight separate CRISPR/guide RNAs in a single experiment.

They have also used the GeneArt Cas9 Nuclease in fertilized zebrafish eggs to create mutant alleles via microinjection of CRISPR/guide RNA stock volume. The team generated a guide RNA to target the tyrosinase gene involved in melanin synthesis. Thus they were able to visualize the impact of the editing, which produced a range of phenotypes with varying mosaic-like levels of melanin migmentation in the body and eyes.

One particular medical application for CRISPR/Cas9 that has generated significant excitement is in the use of stem cells as a source for cell replacement therapies. In a paper published in Cell Stem Cell, a team led by Vijay Sankaran showed that editing hematopoietic stem and pregenitor cells using CRISPR could increase their expansion and differentiation into red blood cells. The researchers employed a clever approach, by using population genetic data to identify mutations associated with increased hemoglobin levels. They identified several rare variants in a gene, SH2B3, that suppressed its function. People with these variants had higher hemoglobin levels compared with controls.

The researchers showed that inactivation of SH2B3 in human embryonic stem cell lines using CRISPR/Cas9 increased red blood cell production more than three-fold. The researchers say the findings demonstrate proof-of-concept that targeting the gene could improve red blood cell differentiation and expansion. With further optimization and cost reductions, it is hoped that in future, engineered stem cells could provide a sustainable source of red blood cells, as an alternative or substitute for donated blood.

References


Conclusion

Since the turn of the millennium we have seen major developments in genome analysis technologies that have, and will continue to, make transformations from basic science through to clinical care.

The arrival of next-generation sequencing has increased the speed of genome sequencing 1000s of times over, multiplying the amount of genetic information we are able to obtain and process. This has already taken centre stage in genetic research and will underpin the coming personalized medicine revolution. In the coming years, more and more people will have their whole genomes sequenced and DNA screening, such as for cancer or prenatally, will take on a greater role in the diagnosis of disease.

Meanwhile, CRISPR gives us a new level of specificity and simplicity in genome editing that one day we will likely see applied to gene therapy. But this is only just one use of this powerful tool, which is already informing discoveries in basic science and drug development, and will also find uses in fields outside of biomedicine, such as environment and agriculture.

At Pittcon 2017, taking place in Chicago from 5-9 March, 2017, you have the opportunity to meet the companies who are providing the tools and technology enabling this new era of genomics. The Pittcon expo will be joined by leaders in the field, including Thermo Fisher Scientific, Eppendorf, Beckman Coulter, Vitl Life Science Solutions, Panasonic Healthcare, and Malvern Instruments.

The conference also hosts The Twenty-Eighth James L Waters Symposium on Genomic Analysis Technologies, which this year will focus on the history, science and technologies of sequencing giant Illumina. We will be fortunate to be joined by co-founder David Walt, as well as former CEO Jay Flatley.

The Symposium will also feature talks Jim Goldberg from genetic screening company Counsyl who will explain how advances in technology have assisted the development of improved screening tools for recessive genetic disorders.

Pittcon 2017 offers a unique opportunity to meet the leading companies and experts who are at the forefront of the genetics revolution. Join us in Chicago in March 2017.