Next generation sequencing (NGS) technology plays a critical role in numerous research fields from unearthing ancient DNA to hunting for novel disease biomarkers. While new NGS library preparation protocols significantly shorten the tedious multiday process of preparing genomic samples for sequencing, many of these workflows still involve monotonous pipetting steps, costly reagents, and substantial hands-on time that delay the path to discovery.
Download this poster from Eppendorf to discover an adaptable platform that scientists can easily modify to meet the unique needs of different NGS library preparation steps and workflows.
Next Generation Discovery: Simplifying NGS Library Preparation
New workflows shorten next generation sequencing (NGS) library preparation and enable more efficient and targeted sequencing. Yet, genomic NGS library preparation still requires multiple pipetting steps, costly reagents, and substantial hands-on time. Automated liquid handling technology alleviates these critical bottlenecks and improves experimental accuracy. Eppendorf®’s epMotion® automated liquid handler offers an adaptable platform that scientists can modify to suit different NGS library preparation steps or workflows. Eppendorf’s team has even assembled an epMotion® NGS bundle, including tube racks, temperaturecontrolled modules, and a plate magnet compatible with most NGS library prep kits that delivers peak performance using validated protocols and Eppendorf application services.
Library Preparation
Fragmentation
The first step in genomic NGS library preparation is enzymatically fragmenting DNA strands into short, uniform pieces. Errors at this stage could lead to incomplete enzyme digestion and improper DNA fragmentation.
End repair and poly(A) tailing
Specialized enzymes then repair DNA overhangs to produce blunt end fragments. The addition of an adenine base, known as a poly(A) tail, to the end of each DNA fragment assists in adapter binding.
Bead cleanup
Magnetic bead-based purification removes stray nucleic acids and other contaminants but requires numerous pipetting steps. Accurate and precise bead handling reduces bead loss and carryover.
Adaptor ligation
Unique sequencing adapters anneal to poly(A) tails of DNA fragment ends. Adding too many or no adapters to sample wells could lead to confounded multiplexed experiments or no indexed sequencing data.
Amplification
Polymerase chain reaction (PCR) amplifies DNA fragments. Smaller volumes and viscous reagents can make it difficult to manually prepare PCR reactions.
Bead cleanup
Double-sided bead purification protocols allow scientists to simultaneously normalize indexed DNA fragments by size and eliminate unwanted fragments and adapter dimers. By changing the bead-to-target-DNA ratio, scientists may selectively purify smaller or larger DNA fragments.
Pooling
Scientists must quantify, dilute, and combine their libraries for accurate equimolar pooling of each fragment. Improper pipetting could lead to uneven sample read distribution and inaccurate results.
Accuracy and precision
Although shortened protocols simplify NGS library preparation, they require precise handling of low sample volumes and accurate reagent dispensing. Automated liquid handlers maintain accuracy and precision across hundreds of samples.
Reliable automation
NGS library preparation entails several sub-steps such as PCR setup, normalization, and bead cleanup that involve tedious and repetitious pipetting. Liquid handlers automate these routine tasks and can even perform more complex workflows, eliminating the risk of human fatigue and error. A built-in optical sensor checks at the start of an automated run that tips, reagents, and labware are in position and liquid levels are correct.
Sample safety
A clean air filter and UV lamp in a closed system maintain a sterile environment. Additionally, non-contact dispensing via automation virtually eliminates cross-contamination.
Intuitive software
epMotion®’s user-friendly software, epBlue, helps researchers easily adjust existing protocols and create new methods. Liquid handlers automate almost the entire NGS library preparation workflow for accurate, precise, and streamlined discovery.