At ASHG 2023, Madan Ambavaram, Ph.D., Senior Applications Scientist at Covaris, discussed the benefits of using Covaris’ Adaptive Focused Acoustics® (AFA®) technology for RNA shearing. He illustrated how this technology streamlines the RNA sequencing workflow, enhancing yield, ensuring better data quality, and providing increased cost savings for libraries, even when using variable samples and ultra-low input.
Introduction
Fueled by advancements in next-generation sequencing (NGS) technologies, RNA-seq studies continue to shed light on the complex world of transcriptomes, providing both quantitative and qualitative insights into the gene expression patterns of prokaryotes and eukaryotes. Central to RNA-Seq lies the fragmentation of RNA and conversion of cDNA, which is generated from RNA samples through a series of steps involving reverse transcription and sample preparation. However, these procedures can introduce biases and artifacts that may pose limitations in certain applications.1
Covaris AFA technology is widely recognized as the gold standard in DNA shearing and has also been extensively used in RNA shearing applications for universal fragmentation that requires little to no optimization and ensures high-quality libraries.
Overcoming Challenges in RNA Sequencing: The Need for Standardization
One of the primary challenges in RNA sequencing is acquiring sufficient high-quality RNA for downstream applications. Variability between samples is a significant concern in laboratory and environmental settings, as is achieving a consistent and specific size distribution of fragmented RNA. The integrity of RNA fragments must be preserved to accurately represent the original RNA sequences. With some methods of fragmentation, mutations or other artifacts may be introduced, and the standardization of RNA lengths can necessitate considerable time and resource investment.
Fragmentation is the initial step in the RNA sequencing workflow. The method chosen for fragmenting RNA can significantly influence the nature of the fragments that are obtained. Many kits available on the market today are based on cation-based RNA fragmentation, using a combination of magnesium and heat. Determining the right concentration of magnesium presents a challenge as both excess and inadequate magnesium can accelerate RNA cleavage by over-riding protection.
Given all of these complexities, RNA experiments require a universal shearing platform that is compatible with different input sample types and concentrations. A streamlined workflow is essential, especially for scalability and adaptability to new kits or sequencing methods.
Figure 1 demonstrates the versatility of Covaris AFA technology, which is agnostic to sample type, for RNA shearing. Whether the RNA originates from cell culture, FFPE tissue, mRNA, or double-stranded RNA, the results remain consistent. The AFA workflow can be fine-tuned to achieve the required fragment size for NGS libraries. Furthermore, scalability is achievable with options for one up to 384 samples in a single run.
Figure 1. RNA shearing using Covaris AFA Technology.
Case Study: RNA Shearing Application with Variable Samples and Ultra-low Input
Recently, the MIT BioMicro Center utilized Covaris technology for shearing FFPE-extracted RNA samples. As illustrated in Figure 2, the top left gel shows the sample quality is highly variable – a common issue with FFPE samples due to their often-fluctuating quality and quantity. The real challenge posed here is how to shear the variable RNA samples with as little as 2 ng of concentration.
Impressively, Illumina libraries utilizing Covaris fragmentation remain notably homogenous, regardless of the variable quality of the input samples, as shown in the bottom left of Figure 2. This indicates that AFA-based fragmentation is unaffected by the input amount, thus ensuring the production of high-quality libraries.
Figure 2. RNA-Seq of Degraded RNA.
In further research conducted by MIT, universal human reference RNA (both intact and degraded aliquots) was used to compare chemical fragmentation and Covaris shearing. As Figure 3 shows, there is an increased log ratio of gene or transcript expression values, along with a consistent mean expression at varying percentages of GC content. This suggests that Covaris shearing delivers consistent and uniform sequencing results, even with degraded samples.
Figure 3. RNA shearing using the Covaris R230 Focused-ultrasonicator.
Advancing mRNA Vaccine and Gene Therapy Platforms with Covaris Shearing
In a collaborative case study with GreenLight Bioscience, Covaris shearing was utilized for their mRNA-based vaccine and gene therapy platforms. The electropherograms depicted in Figure 4 show consistent and reproducible fragmentation — a crucial factor for successful QC and library preparations, especially in high-throughput workflows.
Upon reviewing the sequencing QC data, the team observed more than 99% mapped rates and higher key scores compared to location-based libraries. This led to better coverage, enabling a more comprehensive evaluation of gaps in genome assembly.
Figure 4. mRNA shearing using Covaris AFA.
Greenlight Bio also used Covaris for their double-stranded RNA workflow, with the goal of obtaining Illumina sequencing data from sense and antisense preferential strand sequencing data. The plots in Figure 5 highlight how alternative methods like denaturation showed difficulty in achieving the right fragmentation.
However, using Covaris fragmentation, which requires less optimization time, the team easily achieved the correct fragmentation. This resulted in more than 96-98% of the desired sequencing data. The data reinforces that Covaris AFA shearing delivers reproducible and preferential sense or antisense sequencing data for their applications.
Figure 5. Covaris shearing in dsRNA sequencing workflows.
Conclusion
Covaris AFA technology offers a streamlined RNA sequencing workflow with minimal optimizations that easily meets both current and future high-throughput processing needs. The studies discussed above firmly establish the versatility and efficiency of Covaris’ AFA technology in RNA sequencing applications, catering to low-, mid-, and high-throughput samples.
This unified fragmentation workflow is compatible with a broad spectrum of sample types, including degraded samples, mRNA, or double-stranded RNA, and necessitates minimal to zero optimization. This efficiency translates into considerable cost savings and offers high compatibility with ribosomal RNA depletion, a crucial element in RNA sequencing. Furthermore, Covaris AFA technology stands out by accommodating ultra-low input, permitting the use of samples as minuscule as 2 ng. Therefore, Covaris AFA technology doesn’t just provide solutions; it pushes the boundaries of what’s possible in RNA sequencing.
Reference
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