Ending the runaround with RNA

IPSWICH, Mass. & NORWICH, U.K.—The Earlham Institute, a life-sciences research institute, can sometimes face unique problems raised through the sheer breadth of biological samples that it works with. Samples the institute processes can come from any number of species of plant, animal or microbial organism. Certain plant species can be particularly challenging due to the inherent nature of nucleic acids that are extracted, specifically RNA that is used for next-generation sequencing (NGS) applications. Processing these samples has been problematic, and commercially available approaches have fallen short of producing high-quality libraries from particular species, thus limiting scientists’ ability to study them.

New England Biolabs (NEB) had these specific challenges in mind when they were developing the recently released NEBNext Ultra II Directional RNA Library Prep Kit for Illumina sequencing. “Speaking with scientists around the world, one of the consistent messages we received was that RNA samples continue to present challenges for next-generation sequencing analysis in being of extremely low input and highly sensitive to the variety of contaminants derived from both the organisms themselves, but also the wide range of approaches used to extract RNA from cells,” stated Dr. Fiona Stewart, NEBNext product portfolio manager for NEB.

According to Leah Catchpole, platforms and pipelines team leader at Earlham Institute, the institute heard about the new kit “during a meeting with NEB U.K. NGS application specialist Adam Peltan and Davin Miller, NEB U.K. sales manager, when we were discussing rRNA depletion kits for plants. A need for a robust end-to-end solution for directional RNA-seq was established during these meetings, and we decided to trial NEB’s kit alongside two other competitors. The NEB kit performed best for our diverse set of sample types, so we decided to automate it and bring it into production.”

Says Catchpole, “We trialed NEB’s Ultra II Directional RNA Library Prep Kit using three plant species that have previously been problematic with other RNA-seq kits. We were thrilled with how well and how consistently this kit performed. The NEBNext Poly(A) mRNA Magnetic Isolation Module was also effective, and SortMeRNA showed that we had less than 2 percent contamination for each plant species. The strand specificity was also good at 98 percent.”

“The kit gives very consistent results in rRNA depletion using the Poly A pull down module for all sample types, and strand specificity is also consistently high,” she continues. “Other kits we have trialed have struggled getting consistent results with our diverse set of sample types.”

The Institute is currently in the process of transferring the protocol to their production portfolio as part of the Biotechnology and Biological Science Research Council National Capability in Genomics and Single-Cell Analysis. “It will be offered as a service to our internal science faculty groups and the wider NGS community that we serve,” concludes Catchpole.

In May, NEB announced the expansion of their NEBNext Ultra II NGS library preparation product portfolio to include kits for RNA. The strand-specific NEBNext Ultra II Directional RNA Library Prep Kit and the non-directional NEBNext Ultra II RNA Library Prep Kit are both designed for Illumina NGS systems. The kits are compatible with poly(A) mRNA isolation and ribosomal RNA (rRNA) depletion, and libraries can be constructed from a substantially broader input range (5 ng to 1 µg of total RNA) at high yields. Library complexity and transcript coverage are excellent even at low input amounts and with low-quality samples such as formalin-fixed, paraffin-embedded RNA.

The NEBNext Ultra II workflows include new reagents, combined steps and minimized cleanup steps, for fast and easy use. NEB’s protocols for constructing RNA libraries with Ultra II, including rRNA depletion or poly(A) mRNA enrichment, require under 30 minutes hands-on time and can be finished in approximately six hours. The protocols are compatible with adaptors and primers from NEB or other sources. The workflows are available in kit or module format, and with optional SPRIselect beads for size-selection and cleanup steps.

The NEBNext Ultra II kits address many of the difficulties associated with library preparation, according to reports from early-access users. Dr. Jen Grenier, director of the RNA Sequencing Core at the College of Veterinary Sciences at Cornell University, says, “The Ultra II RNA kit has allowed us to reduce the input for directional polyA+ RNA-seq libraries by a factor of 10 or more. We can now make a library with only 10 ng high-quality total RNA and get the same gene expression profile as for 1 µg input. We’ve even pushed the input as low as 1 ng for very high-quality total RNA. Furthermore, the library prep protocol is streamlined compared to the previous Ultra RNA kits, including a reduction in AMPure bead cleanups and PCR cycles, resulting in better libraries for less time and resources.”