LM_Genomics_eBook_Final GENOMICS RESOURCE GUIDE The tools and technologies that streamline library preparation and sequencing and enhance repeatability AUTOMATION and microplate technology COMPARING sequencing technologies HIGH-THROUGHPUT PCR technology Table of Contents
3 Technological Advances in Genomics 4 Comparing Sequencing Technologies 6 Next-Generation Sequencing: Library Preparation 9 The Latest in High-Throughput Thermal Cyclers 11 Using Microplate Technology to Enhance Next-Generation Sequencing 13 The Benefits of Automated Liquid Handling for Microscale Samples 2 Lab Manager
16 How to Choose the Right PCR Reagents Introduction
Technological Advances in Genomics Labs must weigh factors like library prep and reagents when selecting techniques and technologies for their workflows Next-generation sequencing (NGS) revolutionized the genomics field because of the enormous leaps in throughput and speed, and lower associated costs that make the method more acces- sible. Technological advancements continue to increase efficiency, accuracy, and repeatability across library preparation and sequencing methods. Thith multiple, well-established sequenc- ing technologies to choose from, the decision for labs typically comes down to application requirements, such as sample type, read length, and study design (de novo, shotgun, amplicon, whole genome, etc.), and platform accessibility. Most of the focus for labs is on the more la- bor-intensive library preparation, which is critical to generating high-quality sequencing data. Fortunately, kits, automation, and other supporting equipment streamline workflows for a range of throughput needs, boosting efficiency, accuracy, and repeatability. A range of automat- ed liquid handling solutions from compact pipette stations to full-scale automated workstations and microfluidics instruments can be used to streamline genomics workflows. Automated systems are particularly valuable for ensuring precision and consistency when handling mi- croscale samples common for this type of high-resolution research. Similarly, microplate technology-from the plates themselves to handlers and readers-plays a major role in automation and can have a big impact on sequencing outcomes. Thermal cyclers are crucial for amplification-based sequencing methods and many other workflows in genomics labs. The latest thermal cycler technology includes improvements in speed, capacity, and auto- mation to support DNA amplification, library preparation, and numerous other applications. With ongoing technological advances coupled to reduced costs, the capabilities and applications of genomics research are growing. This resource guide explores the benefits of automation and microplate technology to speed up sequencing and other considerations for library preparation workflows. There is expert guidance on selecting PCR reagents and tips on maintaining automated instruments. The guide also includes a comparison of different sequencing technologies to help identify the best solution for your lab's needs.
Comparing Sequencing Technologies Key technologies meet different sequencing needs by Lab Manager Single-molecule real-time (SMRT) sequencing Uses image-based detection of fluorescently-labeled nucleotides during synthesis. Fragments form circular DNA molecules that are read multiple times to create a circular consensus sequence. Sequencing DNA molecules in real-time by capturing fluorescent signals from nucleotide incorporation Long read lengths, does not require PCR amplification, can detect base modifications relevant to epigenetic studies Can be costly, lower throughput, high error rate (partially mitigated by the consensus sequence formed from multiple sequencing reads per template) DNA fragmentation, adapter ligation Nanopore Detects nucleotide-specific changes in ionic current as DNA molecules pass through nanopores in charged biological or synthetic membranes. Long reads, does not require PCR amplification, can detect base modifications relevant to epigenetic studies, direct RNA sequencing, and well-suited to field use and environmental DNA studies Throughput may be lower compared to other technologies, higher error rates Optional fragmentation or size selection, adapter ligation
Next-Generation Sequencing: Library Preparation Library preparation is a critical step in the workflow of several NGS paradigms By Brandoch Cook, PhD and Rachel Brown, MSc DNA sequencing is perhaps the most substantial develop- ment in molecular biology since the Thatson-Crick structure of the DNA double helix. The earliest method of nucleotide sequencing used chemical cleavage followed by electropho- retic separation of DNA bases. Sanger sequencing improved upon this method by employing primer extension and chain termination, which gained primacy with its decreased reli- ance on toxic and radioactive agents. Since then, pressure on the sequencing data pipeline led quickly to considerable technological changes that far sur- passed the Sanger method in terms of cost and efficiency by flattening the workflow. The high-throughput sequencing methods that followed, collectively known as next generation sequencing (NGS), include several sequencing by synthesis technologies that rapidly identify and record nucleotide binding to complementary strands of amplified DNA, in massively parallel synthesis reactions with a daily through- put in the hundreds of gigabases. Although the principle of massive parallel sequencing reac- tions has been shared across methods, the modes of nucleo- tide incorporation and fluorescence detection in the synthe- sis reactions differ among commercially available platforms. The reagents and library preparation protocols required for sequencing depend on the systems and models used, but some generalities apply. Because of the sensitivity of the technologies and the nature of much modern genomics re- search, success depends on high quality, optimized libraries. Library preparation dictates read depth (number of copies of a given stretch of DNA sequenced), length, and coverage (breadth of sequencing data), which need to be balanced according to the sequencing goals. Greater read depths improve the signal-to-noise ratio and increase confidence in data validity. Regardless of the nature of the starting material-genomic DNA, mRNA, DNA-protein complexes, etc.-the precondition for generating useful NGS datasets is a clean, robust library of nucleic acids. As in so much of molecular biology, there is always a kit for that. A typical, generic workflow for library preparation is as follows: 1) sample collection, fragmentation via enzymatic digestion or shear forces, 2) end-repair and phosphorylation of 5' ends, 3) ligation of oligo dT-based adapters, 4) and a high-fidelity PCR-based amplification step to generate a product with adapters at both ends, barcoded for identifica- tion of individual samples run as multiplex reactions. Most library prep kits are engineered to appropriately modify and amplify the given starting material while reducing the num- ber of steps to accelerate sequencing workflows, maintain sample quality, and minimize contamination. Manufacturers typically provide a wide selection of library preparation and sequencing kits optimized for their platform to suit a variety of applications and sample types. Depending on the se- quencing platform, third party reagents or kits that increase flexibility or reduce cost may be available. Standard library prep kit protocols can usually be performed manually or with varying degrees of automation from com- pact 96- or 384-channel pipette stations to high-throughput, fully automated workstations requiring little to no manual intervention. Automated library prep improves sequenc- ing data by increasing consistency, accuracy, and precision in the pipette-heavy workflows. The boost in accuracy and precision also support the miniaturization of sample volumes, which can be particularly important for low-input sequencing. Sequencing companies often work with multi- ple automation partners to develop validated methods for different platforms. Thhile large robotic workstations are flexible in method development and modifications, they are prohibitively ex- pensive for many labs and best suited for very high through- put environments. Smaller and mid-sized labs that want to take advantage of end-to-end library prep automation that increases walk-away time and reduces the dependency on skilled technicians may have more luck with microfluidics platforms. A shifting technological landscape has produced a variety of self-contained, specialized instruments that produce sequencing-ready libraries post-fragmentation from low to high throughput (starting at around eight samples). However streamlined library prep protocols become, they require a high degree of precision and care to produce reliable sequencing results. Labs have the benefit of many options for methods, kits, and supporting equipment, de- pending on the application, chosen sequencing technology, and degree of automation desired. Product Spotlight Meet the epMotion® 96 Flex Simplify your liquid handling tasks with this versatile semi-automated pipetting system designed to improve efficiency and reliability of your results. What sets it apart? Strengthen your lab's capabilities with the ultimate flex in liquid handling.
CLICK HERE TO LEARN MORE The Latest in High-Throughput Thermal Cyclers PCR-based applications can benefit from the drive toward automation with high-throughput thermal cyclers by Brandoch Cook, PhD The engine driving rapid and predictable temperature changes lies within a circuit board employing Peltier el- ements, the venerable standards for most plate- and plat- form-based thermal cyclers. The Peltier effect occurs when heat is absorbed or emitted at the junction between two dissimilar semiconductors through which a current is passed. A Peltier element is usually incorporated into a conductive metal block, with a ventrally adjacent finned sink through which heat can dissipate. Heating or cooling can be actu- ated and controlled via the amount and directionality of the current. Recent developments in thermal cyclers Newer cyclers feature an increased capacity from a maxi- mum of 384-well to 1536-well plates, a per-assay throughput improvement of four-fold. Commensurate with this increase, per-reaction volumes have decreased substantially, with a maximum of two microliters, versus 10 microliters for the 384-well format. There is throughput, and then there is throughput. Although Peltier elements can deliver predictable and rapid tempera- ture ramps, the constant strain inherent in activating and reversing their potential can cause them to break down over time, and the emitted heat that must be vented can expand the functional footprint of qPCR cyclers well beyond their measured cubic allotment. Alternately, plates can be moved through multi-chambered, water-based thermal cyclers that maintain stable temperatures in discrete baths and thus eliminate by up to 40 percent the time associated with Pelti- er element temperature ramps. Several high-throughput water bath cyclers are available, and can deploy 96-well, 384-well, or 1,536-well plates in rapid succession. Additionally, they can accommodate Array Tape, which obviates the need for plates and can be run almost continuously like newssheet under a printing press because of its low volume and thermal mass. These are endpoint machines optimized for standard PCR. As such, they are primed to take on workflows for large-scale analyses such as mapping genetic interactions. For laboratories looking to automate their genetic analyses, there are attractive options for both real-time and endpoint assays, in configurations suitable for the benchtop. WHEN IT COMEs TO LIQUID HANDLING, AUTOMATION OFFERs sCIENTIsTs A MULTITUDE OF BENEFITs, INCLUDING INCREAsED THROUGHPUT, REDUCED VARIABILITY, FREEING UP TECHNICIAN TIME, AND EFFICIENT UsE OF REAGENTs. ONCE YOU HAVE DECIDED TO sWITCH FROM MANUAL LIQUID HANDLING TO AN AUTOMATED sYsTEM, YOU MUsT FIND A MACHINE THAT sUITs YOUR NEEDs. THEsE TIPs WILL HELP YOU CHOOsE THE RIGHT AUTOMATED LIQUID HANDLING sYsTEM FOR YOUR LAB. PICK A sYsTEM WITH THE RIGHT CAPACITY FOR YOUR NEEDs. CALCULATE THE RUNNING COsTs INVOLVED, INCLUDING CONsUMABLEs. DETERMINE HOW MUCH HANDs-ON TIME Is REQUIRED TO RUN THE sYsTEM. DECIDE HOW MUCH LAB sPACE CAN BE DEVOTED TO THE MACHINE. CONsIDER THE THROUGHPUTs, BATCH sIZEs, VOLUMEs, AND TYPEs OR VIsCOsITIEs OF REAGENTs YOUR LAB WORKs WITH. When it comes to liquid handling, automation offers scientists a multitude of benefits, including increased throughput, reduced variability, freeing up technician time, and efficient use of reagents. Once you have decided to switch from manual liquid handling to an automated system, you must find a machine that suits your needs. These tips will help you choose the right automated liquid handling system for your lab. Download this free infographic, courtesy of
Lab Manager. MAKE A LIsT OF FEATUREs YOU MIGHT WANT TO INTEGRATE WITH YOUR MACHINE, E.G. sHAKERs, INCUBATORs, BARCODE READERs, OR PLATE WAsHERs.
Using Microplate Technology to Enhance Next-Generation Sequencing Plasticware, readers, and other components to speed up NGS by Mike May, PhD Time plays a key role in sequencing. In brief, sequencing sci- entists want results as soon as possible, as long as the results remain accurate. Thith next-generation sequencing (NGS), microplate technology-from the plates themselves to plate handlers and other accessories-impacts many steps in the process. Let's see why these tools still need improvements, as well as the places where microplate technology already enhances the NGS workflow. Microplate technology and supporting instruments have greatly increased sample throughput and reduce the time taken to achieve results in NGS. This said, some challenges remain. Features in need of improvement include uniformity of temperatures and reaction times within each well, vol- umes dispensed and aspirated, dead volumes required, and cross contamination. Technologies related to microplates can also slow down NGS. For example, the slow process of transferring samples into the microplate format. Not all NGS challenges can be solved with better microplates, and a specific lab's needs play a big part in the features that really matter. Improving the parts The miniaturization of reactions in microplates drives the need for automation for accuracy and throughput. This requires mi- croplates specifically designed and optimized for automation. Making such microplates requires application-specific de- cisions from the start, all beginning with developing a mold for manufacturing the plasticware, which might be designed to work with a specific handler. In addition, a manufacturer considers the type of plastic used and treatments to the sur- face. In a microplate designed for NGS applications, a vendor should possess data that validates the expected results. Quantitation also plays a key role in NGS. A robust, sen- sitive, and reproducible quantitation method is required, and the reagents and reader also play important roles. Labs should opt for a microplate reader and reagents with prov- en success for NGS applications, with robust and sensitive detection limits, that are easy to use. In selecting a microplate reader, various features should be considered, including recent improvements. Some of the most exciting advances in microplate reader technology for NGS applications improve the user experience and workflow. Instruments that combine data collection with data analysis can make the user experience and workflow seamless. For example, NGS templates can be quantitated and then auto- matically analyzed for the user, eliminating the need for user input during data analysis. As customers, scientists can seek a complete solution in microplates for NGS-that is, plates made for a specific platform that can automate many NGS steps. That won't necessarily be the least expensive solution, but it could be the best one for a lab that depends on running NGS at the highest possible throughput. THE EVOLUTION of NEXT-GENERATION SEQUENCING From the founding methods to the fourth generation Founding methods Allan Gilbert and Walter Maxam first reported the sequence of 24 base pairs using wandering-spot analysis. DNA was chemically modified, and the DNA backbone was subsequently cleaved at sites adjacent to the modified nucleotides. Frederick Sanger later developed a more efficient technique, the
Sanger sequencing method, and in 1977, published the sequence of a virus genome of more than 5,000 base pairs. The Sanger sequencing method involves synthesizing the DNA that is to be sequenced. Individual synthesis reactions are performed including A, G, C, and T deoxy- nucleotide triphosphates (dNTPs), as well as chain-terminating dideoxynucleotide triphosphates (ddNTPs), which lack a 3' hydroxyl group that prevents phosphodiester bond formation by DNA polymerase and terminates the chain at that position. When synthesis is complete, the products of the individual reactions are separated by size via gel electrophoresis and detected. In the years since the first DNA sequences were obtained in the early 1970s, fluorescence-based sequencing methods have made DNA sequencing easier and much, much, faster. Download the free infographic "The Evolution of Next-Generation Sequencing" courtesy of
Lab Manager. 0 BASE H0 P 0 0 H H 0 dideoxynucleotide
The Benefits of Automated Liquid Handling for Microscale Samples Automating sample handling can fill a growing need for applications including DNA sequencing by Kelsey A. Morrison, PhD To many, the thought of handling microscale samples evokes an image of tedious manual pipetting. This time-consuming task can be largely replaced with automated manipulation of small samples. Automating sample handling can fill a grow- ing need for applications like DNA sequencing. Despite the initial monetary investment necessary to acquire these systems, automated sample handling brings distinct advantages. Laboratories working with small samples by hand face worker fatigue, reduced precision, and limitations on experimental throughput. In contrast, investing in auto- mation can bring obvious benefits, from reduced repetitive motion injuries, greater reproducibility, and increased pro- cessing bandwidth. Additional benefits include savings from fewer wasted samples and reagents, as well as streamlined workflows. The capability to combine sample preparation with analytical instrumentation for fully automated synthe- sis and analysis is another advantage. Common types of systems for handling small-scale samples Likely the most recognizable form of automatic sample handling, pipette-based systems act as robotic pipetting plat- forms by dispensing solution from tips through contacting the deposition target. These pipette-based systems typically operate through either an air-cushion design for sample manipulation or with positive-displacement via pistons. For applications requiring higher accuracy and precision of low-volume samples, positive-displacement is preferable over the lower cost, lower precision air-cushion mechanism. Similar to the pipette-based sample handling systems are those based on syringes and pins, both of which require contact between the dispensing device and intended end surface or solution. All three forms of the contact-based liquid manipulation platforms have the potential drawback of cross-contamination. For laboratories that can afford to invest in an automated sample handling platform based on mechanisms other than pipetting, syringes, or pin dispensing, the alternatives may be better options when high precision and accuracy are para- mount, if low- and sub-nanoliter samples are to be processed, or cross-contamination is a concern. A direct comparison of results based upon data collected from samples handled in a tip-based system and in an acoustic droplet ejection (ADE) platform found statistically different results between both datasets, with the ADE system appearing to provide more consistent values. ADE sample handling is also useful for rapid, microscale synthetic prototyping, which is how it was applied for automatic reaction scouting of isoquinoline synthetic building blocks in nanoliter droplets. Microscale acoustic manipulation has a wide range of potential appli- cations because of its precise control, short dispensing time, and compatibility with high-capacity sample wells rendering the mechanism particularly appealing in bioassays. Other forms of non-contact, high precision liquid handling are systems employing microfluidics, solenoid microvalves, and piezoelectric devices for aliquot ejection as some of the major classes of liquid manipulation technologies. Beyond simply a liquid transfer device, automation of sample han- dling with microfluidics offers the possibility of higher-order sample preparation, such as sample mixing, separations, and other preparatory steps for small sample sizes. Automated liquid handling with solenoid and piezoelectric devices has demonstrated accuracy and precision that is highly suitable for sensitive assays, even for picoliter and nanoliter volumes. 14 Lab Manager Genomics Resource Guide Maintaining Automated Liquid Handlers Liquid handlers require regular maintenance for consistent, reliable results By Sachin Rawat Most operations in a laboratory involve moving around various amounts of liquids. While manually doing this is practical for smaller experiments, biology is increasingly high throughput. DNA/RNA extraction, fragmentation, and library construction and other applications can be streamlined and standardized with automated liquid handling. Unlike manual pipetting, automated liquid handling systems can do this quickly, are cost-efficient, and offer high repeatability and precision. Common problems with automated liquid handlers: Best practices for maintaining automated liquid handlers:
How to Choose the Right PCR Reagents Dr. Gregory Shipley, director of the Quantitative Genomics Core Laboratory at The University of Texas Health Science Center in Houston, discusses how he goes about evaluating and investing in new technologies and reagents by Lab Manager Q: What would you advise for lab managers who have to continuously keep up with the new reagents and instrumentation being introduced into the market? A: If you see a new reagent, I would suggest first talking to the salesperson and getting the person to send you a small aliquot to try. Then do some side-by-side comparisons with your old and new reagents, using the standards that you use for all your assays, and see if the new reagent works better or at least as well. If it has a better price, then it's worth con- sidering. The ready-made kits are a good deal because they save you time, but they cost more. So if you are operating small, then you may want to buy individual reagents and save money by making your own master mixes. However, if you are brand new to the game, then I highly recommend that you start with a kit, because you don't want something going wrong because of the reagents. Q: What about when evaluating new instrumentation or software products? A: It's the same thing. If you are serious about buying new equipment, you can get the company to bring in the ma- chine for a week or so, to see how it works. If it doesn't work for you or it doesn't do what you need it to do, don't buy it. Don't buy a machine for what you need now, but buy it for what you think you might need in the future. If you buy an inexpensive machine or don't buy the various software modules, then you may not be able to do certain things like running 384-well plates or multiplexing or HRM (high-reso- lution melt). Sometimes people buy new PCR machines with a certain project in mind, but what you really have to do is think ahead. Q: What advice do you have for labs looking to negotiate a better price with various vendors? A: Things have changed a lot in recent years. For instance, The University of Texas system has negotiated a certain price with vendors, and so every lab on our campus gets that same price. Other places are doing the same by leveraging their buying power. However, if you are in a small lab but can show that you are using a lot of reagents or if you can pool together with other labs to show purchasing power, then you can negotiate a better price. There are also "freezer pro- grams" set up in many institutions where scientists can walk to and pick up reagents directly from the vendor-installed freezers or refrigerators, which saves money by eliminating shipping costs. Q: What kinds of challenges do you run into when working with PCR reagents and how have you managed to overcome them? A: Part of what you are paying for with the ready-made reagents is the quality control that goes into the reagents before they come into the market. The same goes for other PCR reagents like primers, vectors and probes. One thing, however, that I would like to point out is that, although the companies that make oligonucleotides do their best to tell you how much reagent is in the tube, sometimes the amount varies. Hence, we always determine the exact amount of what is in the tube by looking at the optical density of the primers and probes. There is always some loss, especially af- ter the reagent is dried down in the tube, and every now and then we get something that is almost tenfold less than what is expected. This is happening less and less than it used to, but, nonetheless, what's on the label should be used only as a guide. There are also some problems with contamination. For instance, every primer and probe has some slight DNA contamination and you can pick it up with the assays that are really sensitive. But there is nothing you can really do about it. Fortunately, it isn't a problem for the vast majority of assays. Eppendorf is a leading life science company that develops and supplies products catering to academic and commercial research laboratories. The product portfolio is made up of instruments, consumables, and services for liquid, sample, and cell handling. For over 75 years our products have gained the trust of generations of laboratory researchers. Our scientists and engineers are on a constant mission to build on trusted methods to help address the challenges that today's scientists face. Building on a foundation of collective experience, history and knowledge, we are committed to the future and continuously strive to improve human living conditions.
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