With the growing appreciation that microbes affect health in ways other than simply causing infections, academia and industry are forming collaborations to advance knowledge and to set the stage for microbiome-based treatments and diagnostics.
To this end, workflow standardization from sample collection and storage through nucleic acid purification and next-generation sequencing (NGS) library preparation is critical in order to compare and combine separate studies in consortium-based approaches at a global scale. Eppendorf, a leading life science company, provides a range of products to support this effort.
According to a recent review published by Rob Knight and colleagues,1 scientific study of the microbiome, especially case–control and longitudinal studies, requires best practices and reproducible laboratory workflows. Hundreds, sometimes thousands, of samples are processed for the identification and comparison of microbial community structure, composition, and genetics, as well as functional variation.
To avoid overrepresentation of easy-to-culture species and give an unbiased picture of genetic diversity, NGS and other molecular techniques can use freshly extracted nucleic acids without the need for culture. The entire genome of a species can be studied, or specific regions of the genome selected to help species identification (for bacteria and archaea, the 16S rRNA region is often used) and enable faster and more efficient microbiome analysis.
The importance of standardization
From the start, it is crucial to avoid variation during sample collection and shipping, and to allow for storage at –80°C. The high capacity per footprint Eppendorf ULT Freezer line ensures temperature stability, fast recovery times and integrates easily with eLABinventory, the company’s sample management software and VisioNize remote device monitoring software.
Samples can vary, and can contain different concentrations of microbes, can be heavily contaminated with relic DNA, and require special processes. Accordingly, the usage of established extraction and NGS library preparation procedures are very important. This involves turning freshly isolated nucleic acids into fully prepared, bitesize chunks that are ready for sequencing.
Library preparation steps, including DNA extraction, quantification, fragmentation, normalization, purification and QC pose major labor-intensive bottle necks for the generation of sequencing data. In particular, the high number of liquid-handling operations in sequencing sample preparation and standardization is challenging—especially in microbiome research that involves working with many different species. With more liquid handling the risk of errors increases, which leads to outliers and inconsistent sample quality and yields that affect the reliability of the entire study.
Repeated pipetting, mixing and separation are all well suited to automation to eliminate human errors and ensure high accuracy, even at small sub-microliter volumes. Standardization helps to keep the error rate low as the throughput goes up. As a result, the use of automated liquid handling technology improves accuracy and consistency in library preparation freeing time for data analysis.
Eppendorf offers many products to support different aspects of sequencing workflows. The multi-purpose liquid handling workstations, epMotion® 5073m and 5075t, are easily integrated into many Illumina, KAPA and NEB workflows—bringing accuracy, consistency and ease of use to liquid handling for high-quality NGS libraries. The on-deck thermomixer provides needed incubation capability.
The new small footprint epMotion 5073m NGS solution is ideal for small-scale library preparation of up to 24 samples or magnetic bead based DNA purification from 96 samples, even single tubes can be accommodated; whereas the epMotion 5075t accommodates up to 96 samples for both procedures.
The automated workflows allow the generation of ready-to-sequence libraries with minimal setup time. Adapting state-of-the-art kits from a manual to an automated process is straightforward, and Eppendorf has many qualified protocols available. In addition to the liquid handlers, the company offers LoBind® consumables and the MasterCycler® X50 PCR cycler to maximize library yield and quality.
Standardization and automation will continue to play increasingly important roles to ensure reproducibility in microbiome analysis.
1. Knight, R. et al. Nature Rev. Microbiol. 16, 410-422 (2018).