Expanding the Short-Read Sequencing Space With Novel RNA-Sequencing Library Preparation Workflows

New short-read sequencers promise increased access to RNA sequencing

With the introduction of new short-read sequencing platforms, the contest to disrupt the next-generation sequencing (NGS) market has begun, with rapid, accurate and affordable sequencers providing alternatives to the leading commercial “sequencing-by-synthesis” instrument.  As a result, researchers can now choose from these new platforms for analyzing the expression of RNA transcripts accurately during RNA sequencing (RNA-seq). 

Here we show how improved access to RNA-seq can fulfill its potential benefits to translational research and summarize how novel RNA library preparation chemistries can enable the acquisition of robust data from these new sequencing platforms, and profile a range of sample types, with thoughts on further areas of innovation.

The benefits of RNA-seq can provide utility in many application areas. Broader adoption of RNA-seq in basic science research could lead to better annotation of the genome and therefore an improved understanding of the functions of many new genes.

Findings from RNA-seq are also showing unique clinical advantages, specifically in detecting biomarkers and profiling tumor protein expression in formalin-fixed paraffin-embedded (FFPE) samples, which are arguably the most common archived biospecimen available. In a recent study, detection of brain tumor gene fusions in FFPE samples by next-generation mRNA sequencing led to decisive diagnostic relevance in 43% of cases, while in 40% of the cases, the detection of fusion genes provided a druggable target for those patients.

Given limitations in the diagnosis and treatment of brain tumors, such outcomes are notably significant. Such advances are not limited to brain tumors. For example, RNA-seq clinically applied to breast cancer enables the prediction of prognosis and other outcomes. Enabling more discoveries like these requires the introduction of more accessible platforms that make RNA-seq available to a broader number of researchers. Given improved accessibility, these new platforms could aid in the potential development of new discoveries, or empower clinical sequencing applications for predictive diagnostics.

The role of RNA sequencing library preparation in technology disruption

Ensuring these potential uses of new short-read sequencing platforms goes beyond the instrument purchase price, RNA isolation and sequencing reagents. It also requires RNA library preparation reagents that enable high-quality sequencing outcomes for a broad range of sample conditions. 

Library preparation is a key step in RNA-seq and involves the conversion of RNA to cDNA. An adapter ligation step introduces a sequence to the 3’ end of the RNA transcript for use by the reverse transcriptase during first-strand cDNA synthesis. Subsequently, amplification, size selection of specific fragments and clean-up occurs, after which the cDNA library can be run on the sequencer. Following the sequencing run, read lengths of 50–300 base pairs (bp) are analyzed separately and assembled into a whole genome sequence using open-access scripts during bioinformatic analysis. To identify the transcripts detected, the analysis compares the data pipeline to a reference genome for that species.  

RNA sequencing needs to be suitable for a range of sample types to ensure broad adoption. While many library preparation options are available for fresh samples and isolated cell cultures; fixed tissues, such as FFPE samples, remain a challenge for the high RNA quality requirements of many commercial RNA-seq library prep kits. RNA isolated from FFPE tends to be of poor quality as it is often heavily degraded and chemically modified from crosslinking during fixation. Given that the vast majority of tumor biopsies are stored as FFPE, the ability to use RNA from fixed tissues for sequencing would allow greater access to sequencing data from the most common archived specimen type. As a result, developing protocols for FFPE samples would enable the use of numerous sample archives for valuable clinical research.

Methods enabling whole transcriptome analysis of FFPE samples

To address the challenges of FFPE, a recent study evaluated the SEQuoia Complete Stranded RNA library prep kit for FFPE against a leading commercial kit, for the capture of the whole transcriptome, including both long and short RNA biotypes, in breast cancer samples. The SEQuoia workflow used the SEQuoia Complete Kit followed by a novel post-library method to remove the ribosomal RNA-derived cDNA fragments with the SEQuoia RiboDepletion Kit. The group identified that RNA-seq library preparation using the SEQuoia workflow could rapidly provide both short and long RNAs from a single workflow, overcoming the biases inherent to the other commercial kit, which only captures the specific size range of long RNAs. For research where speed and ease are prioritized, the SEQuoia Express Stranded RNA Library Prep Kit offers a rapid 3-hour, 3-tube workflow that detects long RNAs from higher-quality RNA samples.

The SEQuoia Complete and SEQuoia Express Kits eliminate common challenges associated with RNA-seq by first eliminating bias introduced by typical multistep enzymatic processes. Using a single engineered retrotransposon (i.e., SEQzyme in the SEQuoia workflow), in place of the traditional reverse transcriptase and ligases found in standard pre-libary ribo-depletion strategies eliminates the bias resulting from a multi-step enzymatic process, and produces cDNA libraries which more closely resemble the source transcriptome. Additionally, SEQzyme enables improved processivity and end-to-end template jumping by using a continuous synthesis reaction that converts RNA to cDNA and adds both sequencing adapters in a single step. Moreover, in the SEQuoia Complete Kit format, the RNA fragments are first polyadenylated and then primed with a polythymidine sequence on the 3’ end of the sequencing adapter. As a result, both long (>200 bp) and short (<200 bp) RNA transcripts can be captured in one library workflow.

A second advantage of the SEQuoia workflow is that the post-library ribodepletion strategy preserves transcriptome richness by depleting ribosomal RNA after its conversion to cDNA, at a point when the sample is more stable. These unique attributes ensured a more robust process leading to more complete transcriptome profiling.

Overall, this work shows the promise of RNA-seq to unlock novel molecular mechanisms, so that in future, biologically-driven studies can occur even with FFPE and other degraded and/or low yield RNA samples.

Further innovation is needed to address RNA sequencing challenges

While new sequencing platforms and robust reagents are driving the adoption of applications that utilize RNA-seq technology, many other aspects upstream and downstream of this process require innovation. RNA-Seq is also part of a larger molecular analysis workflow. For example, pairing RNA-seq with digital PCR and qPCR allows the confirmation of a broader discovery process using discrete smaller panels that are quick to implement in a large group of samples, once the relevant targets for a biological process are identified.  

Key areas where commercial focus could benefit or even transform RNA sequencing workflows include:

• Eliminate bioinformatics bottlenecks by providing integrated computational tools and resources aligned with new sequencing technologies for small and long RNAs
• Improved RNA extraction reagents which ensure stable, high-quality RNA at sufficient levels of yield would reduce the risk of degraded RNA from sample handling. 
• Sample pooling to run up to 96 libraries in a single tube to dramatically improve capacity
• Automated and streamlined RNA-seq library preparation for higher throughput and standardization
• RNA-seq data confirmation using orthogonal molecular technologies, such as digital PCR and qPCR 
• Expansion of reference genomes for non-mammalian species, such as common crop plants and pathogens
• Overcome the affordability barrier to greater sequencing depth in the billions — dramatically increasing the number of reads per sample could identify low-level, previously undetectable transcripts
• Using single-cell RNA-seq to determine cell-type-specific outcomes in the diverse heterogeneous tissue microenvironment aids developmental biology, neuroscience and oncology
• The development of robust, cost-effective long-read sequencers capable of handling large fragments up to 1000 bp or more, to evaluate more of the RNA transcriptome, and fusion proteins, which short-read sequencers cannot detect.

It is also essential to ensure that RNA-seq library preparation kits are readily suited to transcriptome profiling for a range of sample types with varying RNA input, and are compatible with new short-read sequencing platforms, allowing the transfer of existing workflows to these new instruments or the development of new ones. For example, recent studies have been undertaken to demonstrate the compatibility of the novel RNA-seq library preparation discussed above with new sequencer platforms (Figure 1 + 2), including Element Biosciences’ AVITI platform and the Singular Genomics’ G4 system.

It is also essential to ensure that RNA-seq library preparation kits are readily suited to transcriptome profiling for a range of sample types with varying RNA input, and are compatible with new short-read sequencing platforms, allowing the transfer of existing workflows to these new instruments or the development of new ones. For example, recent studies have been undertaken to demonstrate the compatibility of the novel RNA-Seq library preparation discussed above with new sequencer platforms (Figure 1 and 2), including Element Biosciences’ AVITI platform and the Singular Genomics’ G4 system.

Figure 1: SEQuoia Complete libraries run on the Element AVITI System produced consistent sequencing results in long RNA transcript detection among sample replicates and different RNA input levels (left) and also detected small RNA transcripts with high (100 ng) or low (10 ng) RNA input (right). Credit: Bio-Rad.

Figure 2: SEQuoia Express Stranded RNA libraries run on the Singular G4 provide detection of more long RNA transcripts compared to leading commercial benchtop sequencers, for both high (100 ng) and low (10 ng) RNA input. Credit: Bio-Rad.

Through this strategy, not only can users bridge the gap to more affordable sequencing, but they can also obtain robust data which rivals the leading commercial benchtop sequencing platform. Depending on the kit used, researchers can obtain the full transcriptome profile from their samples at lower cost and effort, and gain access to high-quality data even if highly degraded RNA from FFPE samples is used. Or if speed and convenience is a key consideration, using a more rapid library prep workflow can ensure that work is completed in under 4 hours.

Excellent library preparation is key to RNA sequencing advances

RNA-seq has revolutionized the era of RNA analysis in less than a decade, allowing research to forge into the unknown aspects of the transcriptome and delineate fundamental biological processes and disease states. Improving access to this unique technology in the next decade will help realize its full potential in both biological and translational research.

About the authors

Nish Kumar is senior product manager for the Global Gene Expression Group at Bio-Rad. She has a strong genomics background and works on new product development ideas.

Linda Lingelbach is the marketing manager for the Global Gene Expression Marketing Group at Bio-Rad, responsible for bringing novel NGS library prep kits and qPCR consumables to market so researchers can accomplish more with their research.

Angelica Olcott is the senior applications manager for the Global Gene Expression Marketing Group at Bio-Rad. She enjoys supporting marketing efforts to create engaging technical content and working with key researchers in the areas of RNA-Sequencing library preparation and multiplexed qPCR.

Cissy Jiang is a senior global product manager leading the commercial product marketing efforts for Bio-Rad laboratories' RNA sequencing library prep product portfolio. She shares a passion for technology innovation in gene expression analysis and strives to enable Bio-Rad’s revolutionary products in the NGS arena.