The last decade has seen an explosion in genomic medicine, which has revolutionized the diagnosis and care of individuals with genetic disorders, cancer, and infectious disease. Instead of waiting an average of eight years for a diagnosis, a patient with a rare genetic condition may wait only days or hours. Cancer patients who previously only had months to live, may survive a decade longer because of tailored and targeted interventions. The lives of individuals with enigmatic infections are routinely saved because of the rapid identification of a pathogen.
This is an exciting time for biological research. Meaningful discovery is churning at a pace unmatched throughout history. Thanks to the pioneering world of next-generation sequencing (NGS), the data we can derive from organisms is deeper and more insightful than ever imagined. NGS equipment, once reserved for the largest and busiest of research centers, is now accessible to enterprises of all sizes – unearthing new knowledge on cancer, microbiology, genetic disease, reproductive health, agriculture and forensics, and other emerging areas.
NGS in lab space
With the advent of the first FDA-approved NGS instrument in late 2013, the technology is now gaining a stronger foothold in the clinical laboratory space for screening and diagnostic testing. Whether you are seeking to conduct research worthy of publication in a respected journal or looking for critical clues to the spread and evolution of disease, your work likely requires or would benefit from NGS technology. For this reason, labs that do not already own an NGS system are making a push to acquire equipment of their own. For many organizations, it’s a matter of data privacy or a need for tight control over their projects and samples. For others, it’s simply an issue of efficiency and timesaving. For all, it’s seen as an investment in high-quality, reproducible data that leads to valuable biological insight.
If you are a principal investigator or manager for one of these labs, you have an important purchasing decision to make. The good news is that the market today offers plenty of choice. Next-generation systems range from ultra-flexible, high-output instruments that can read multiple samples in a single run, to desktop sequencers ideal for smaller-scale studies. This guide will walk you through the purchasing process by helping you evaluate your research goals and laboratory needs.
First, let’s tackle some of the most important questions that will define your product search: What types of NGS experiments do you plan to perform? And which applications will you use most? Think broadly here, considering your current needs as well as needs in the future. Purchasing NGS instrumentation is a commitment, and you want to be sure that your chosen instrument will provide the versatility and the power to accommodate your lab for years to come. For each application, consider your needs for these three areas:
Throughput per run
- Your needs in this area will largely dictate the type of sequencer that is best suited for you. Assess your projected sample numbers per month and year, and let this number guide your selection process. For example, if you plan to run large sample numbers for applications such as whole-genome or whole transcriptome sequencing, you are probably a good fit for a high-throughput system.
Read length
- Longer read lengths are very important for many applications, with de novo sequencing standing out as a prime example. Why? The longer the read, the better the coverage across the genome. And because de novo sequencing does not use a reference genome to align data, it is essential to produce long overlapping reads to complete the assembly. Applications such as microbial sequencing need long reads, as many viruses and other microorganisms have not been previously sequenced. Large structural chromosomal rearrangements in cancer genomes can also be difficult to detect without long reads, as the reads must span the chromosomal breakpoints in order to detect them.
Paired-end sequencing
- A broad range of applications can benefit from paired-end sequencing, which allows for maximum coverage across the entire genome. If you currently require or will require paired-end sequencing, be sure to investigate the solutions available for each of the instruments you are considering. It is important that the equipment supplier has established protocols and produces high-quality data with this technique. Easy sample preparation and data analysis tools are also valuable in this area, as a simple and integrated workflow will save you time and money.
Think about budget from DNA to data
Your budget can help you narrow your decision on the type of sequencer to purchase, but the issue of investment is not as clear-cut as you might think. There are many important factors to consider beyond the initial capital expenditure of the instrument itself, including the cost of ongoing operations, the quality of the data (ensuring the process won’t have to be repeated at an additional cost) and the hands-on time required to get the job done right.
This is where it is critical to consider your research needs, as well as the labor required to prepare samples for sequencing and to analyze your results. What reagents will you need on an ongoing basis? How long will it take to interpret the data? How and where will you store the data? These are some of the most important issues to consider. Examine your budget per sample and make sure everything is included in that cost, including DNA extraction and informatics.
Operational expenses: cost per sample
Consumables are one of the biggest drivers of operational expenses, as they are required for both library preparation and sequencing. Library prep costs will vary depending on the study size, application, and vendor. Many third-party vendors sell library prep kits, but not all kits will work on every machine. That is why it’s smart to select a sequencing technology for which various kits are sold. This ensures a larger range of options and, oftentimes, more competitive prices. When comparing operational costs, look at the cost per base or cost per sample. This figure is dictated by the amount of DNA that can be sequenced per run. For higher-output applications, economies of scale can help reduce cost. Tip: Sample indexing allows you to pack more samples into each sequencing run, lowering the cost per base. For labs running high sample numbers, the higher expense of a high-throughput sequencer is offset over time by the lower operating costs associated with each sample.
Hands-on labor: time is money
Another factor to consider with operational costs is the hands-on time required by lab technicians. The more time a lab tech must spend to carry out a given sequencing experiment, the less time that person has available for other important projects. It becomes an issue of both money and time. Consider the element of efficiency as you compare platforms. Tip: In addition to hands-on time, pay attention to the amount of user intervention needed per experiment. Even if hands-on time is minimal, you may have to return to the equipment often to intervene. This cuts into a user’s ability to do other things in the lab, and, as a result, increases the operational costs.
Envision new workflow and informatics
You want your NGS system to foster the easiest workflow possible. Considering the scope of many DNA experiments, you should seek out any and all opportunities to save time and ensure accuracy. The quantity of data NGS produces can seem overwhelming at first. Next-generation sequencing data output has increased at a rate that surpasses Moore’s law, more than doubling each year since it was invented. In 2007, a single sequencing run could produce a maximum of around one gigabase of data. Today, that rate exceeds a terabase of data in a single sequencing run — more than a 1,000x increase.
What does this mean for you? For starters, it means that you’ll have to find a way to manage a very large amount of data. So, as you size up your NGS options, think about what each system will mean for your workflow, from sample preparation to informatics. How exactly will you turn that raw data into actionable information? Make sure you are comfortable with the workflow, not intimidated by it.
Sample and library preparation
Consider the types of experiments you do and how many samples they usually involve. When evaluating your NGS options, look specifically at how many days it will take to create the libraries. How much of that time is “hands-on” time? Does the equipment vendor offer a library solution? In general, sample preparation protocols for NGS are more rapid and straightforward than those for Sanger sequencing.
With NGS, you can start directly from a gDNA or cDNA library. The DNA fragments are then ligated to platform-specific oligonucleotide adapters to perform the sequencing biochemistry, requiring as little as 90 minutes to complete. When considering sample preparation, ask vendors about the versatility of their products: Do the sample prep solutions support a broad range of applications? Is it easy to purchase all of your needed solutions in one place and access support when questions arise?
Data analysis
Along with sample preparation, data analysis is an area that simply cannot be overlooked when comparing NGS equipment. A wide range of data-analysis algorithms are available that perform specific tasks related to a given application. Some applications require specialized assembly of sequencing reads.
Others require quantification of read counts to provide information about gene expression levels. While some of these data analysis applications are commercially available from software vendors, many are freely available opensource algorithms from academic institutions. Explore what types of analysis tools and protocols are available to users of a given platform and ask whether these tools are specialized in working with data from the equipment you are looking to purchase. Ease of workflow and data integrity are at stake. It is even better if some tools are open source because users and developers continually make improvements.
Conclusion
Revolutions in diagnostics, precision drug development, population health, forensics and environmental science have all been underpinned by NGS technology; and we are on the cusp of making genomic medicine a reality for hundreds of millions across the globe. In the span of just over 20 years, genomics has upended the paradigm of the slow introduction of new technologies into medicine and has the potential to further disrupt how the world thinks about their health care. As we look to the future, genome sequencing for every child at birth, every patient with rare disease or cancer and any individual with concerns about reproduction or long-term health risks is within reach.
(This article was created with the permission of Illumina, a provider of next-generation sequencing technology. For more information, visit www.illumina.com).
