Novel, high-throughput 3’ mRNA-seq technologies like MERCURIUS™ BRB-seq from Alithea Genomics and the QuantSeq-Pool and QuantSeq 3’ mRNA-seq library prep kits from Lexogen are driving cost-efficient, transcriptomic experiments at a larger-scale than ever before.
In this article, we compare the workflows of each of these technologies.
Overview of MERCURIUS™ BRB-seq and QuantSeq technologies
MERCURIUS™ BRB-seq from Alithea Genomics and QuantSeq technologies from Lexogen each add barcodes to the 3’ end of mRNA molecules. This approach removes the need for sequencing the whole length of a transcript. It allows researchers to sequence more samples at a lower read-depth, with no loss in data quality compared to traditional RNA-seq methods such as Illumina TruSeq library preparations (Alpern et al., 2019).
The three 3’ mRNA-seq technologies are similar but the QuantSeq 3’ mRNA-seq library prep kit requires the researcher to process each sample individually throughout the whole protocol.
Conversely, MERCURIUS™ BRB-seq and QuantSeq-Pool kits allow researchers to pool samples early in the workflow and identify each sample during final data analysis. This early multiplexing streamlines subsequent steps and reduces technical variation as all samples are prepared simultaneously in the same tube.
Overview of multiplexing capacities
Each 3’ mRNA-seq kit differs in the number of samples that researchers can multiplex in one experiment.
QuantSeq-Pool has the largest capacity for multiplexing. Researchers can include 384 Illumina i5 and i7 unique dual indices to allow multiplexing of up to 36,864 samples.
QuantSeq 3’ mRNA-seq library prep kits use a very similar workflow to QuantSeq-Pool. It includes an optional 96 i7 indices but, when combined with 96 i5 indices, up to 9,216 samples can be multiplexed.
MERCURIUS™ BRB-seq allows for up to 1,536 samples to be multiplexed as standard thanks to combinations of i5 and i7 indices. However, multiplexing is highly flexible with larger-scale customized options available.
Comparison of workflows
The first step in each kit is to add unique oligo(dT) barcoding primers to total RNA from a specific sample in each well of a 96 or 384 well plate. Alithea Genomics and Lexogen technologies use different barcoding sequences and lengths but the overall components are similar.
Both MERCURIUS™ BRB-seq and QuantSeq-Pool include an adaptor for primer annealing, a 12 to 14-nucleotide long sample barcode that assigns a unique sample identifier to each RNA sample, and a 10 to 14-nucleotide long unique molecular identifier (UMI). The UMI distinguishes between original mRNA transcripts and duplicates that result from PCR amplification.
In QuantSeq 3’ mRNA-seq library prep the inclusion of the UMI sequences is optional and they are added at the second strand synthesis step.
Next, mRNA is reverse transcribed into single stranded cDNA. Each technology performs the reverse transcription step via oligo(dT) priming.
At this stage, the MERCURIUS™ BRB-seq and QuantSeq-Pool methods pool all samples from the 96 or 384 well plate into a single tube to streamline subsequent steps.
In the QuantSeq 3’ mRNA-seq library prep kit each sample is processed individually and not pooled.
MERCURIUS™ BRB-seq uses DNA pol I-mediated nick translation for second strand synthesis, whereas the Lexogen QuantSeq-Pool and QuantSeq 3’ mRNA-seq library prep technologies use random priming.
Full length cDNA is then ‘tagmented’ with Illumina specific index sequences for library amplification. The index combinations used depend on how many samples the researcher needs to multiplex.
Why choose MERCURIUS™ BRB-seq over QuantSeq technologies?
In a direct comparison between MERCURIUS™ BRB-seq and QuantSeq-Pool under the same conditions, MERCURIUS™ BRB-seq had a higher demultiplexing rate, higher number of uniquely mapped reads, and detected almost twice the number of differentially expressed genes compared to QuantSeq-Pool.
Therefore, MERCURIUS™ BRB-seq from Alithea Genomics performed better than the Lexogen QuantSeq technologies under the same conditions, with a lower cost per sample.
To find out more about how MERCURIUS™ BRB-seq can help in your study, please contact us at info@alitheagenomics.com.
References
Alpern, D., Gardeux, V., Russeil, J., Mangeat, B., Meireles-Filho, A.C., Breysse, R., Hacker, D. and Deplancke, B., 2019. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome biology, 20(1), pp.1-15.
