Cost-effective 3’ mRNA-seq technologies: How similar are they?

Novel, high-throughput 3’ mRNA-seq technologies are driving cost-efficient, transcriptomic experiments at a larger-scale than ever before. Numerous 3’ mRNA-seq options are now available, such as the MERCURIUS™ BRB-seq kit from Alithea Genomics, QuantSeq-Pool from Lexogen, and the QIAseq UPX 3’ transcriptome kit from QIAGEN.

All three 3’ mRNA-seq technologies rely on sample barcoding primers. Barcoding allows researchers to pool samples into the same tube early in an experiment. This reduces technical variation, hands-on-time, and increases the cost-effectiveness of 3’ mRNA-seq experiments.

In this article, we compare the specifications of these different 3’ mRNA-seq kits, including their overall method, hands-on-time, the volume of input RNA needed, and their potential to multiplex samples.

Comparison of 3’ mRNA-seq methods

The first step in each of the 3’ mRNA-seq methods is to add unique barcoding primers to total RNA from a specific sample in each well of a 96 or 384 well plate. The sequences used for barcoding differ for each method.

These barcodes tag the mRNA in each well for sample identification at the analysis stage. They also contain a unique molecular identifier (UMI) to determine PCR duplicates during final analysis, as well as index sequences necessary for Illumina sequencing platforms.

Next, barcoded mRNA is reverse-transcribed into single-stranded cDNA. QuantSeq-Pool and MERCURIUS™ BRB-seq perform the reverse transcription step via oligo(dT) priming, whereas the QIAseq UPX 3’ transcriptome kit uses oligo(dT) priming alongside a template switching oligo.

At this stage, each method pools all samples from the 96 well plate into a single tube.

The next step is second strand synthesis. Each kit uses a different method for this process. The QIAseq UPX 3’ transcriptome kit requires PCR amplification. However, excessive cycles of PCR generally reduce the quality of data output.

Both the MERCURIUS™ BRB-seq and QuantSeq-Pool methods avoid this PCR step. Instead, they use DNA pol I-mediated nick translation or random priming for second-strand synthesis. Full-length cDNA is then ‘tagmented’ with Illumina-specific index sequences for library amplification and multiplexing.

Potential for multiplexing

QuantSeq-Pool has the largest capacity for multiplexing. Optional inclusion of 384 Illumina i5 and i7 unique dual indices allows 36,864 samples to be multiplexed.

For the QIAseq UPX 3’ transcriptome kit, researchers can multiplex 18,432 samples for sequencing in a single Illumina flow cell lane.

MERCURIUS™ BRB-seq allows for 1,536 samples to be multiplexed but with larger-scale, customized options are available.

Comparison of hands-on-time

The amount of time required to perform each experiment is also an important consideration. QuantSeq-Pool has the shortest hands-on-time of 1.5 hours, MERCURIUS™ BRB-seq has a hands-on-time of approximately 2.75 hours, whereas QIAseq UPX 3’ transcriptome kit has a longer hands-on-time of up to one day.

Comparison of recommended RNA quantity

For QuantSeq-Pool, the recommended input amount of total RNA is 10 ng per sample. The MERCURIUS™ BRB-seq kit also recommends between 10 – 1000 ng of per sample. The QIAseq UPX 3’ transcriptome kit recommends different amounts of RNA depending on the numbers of reads required. It recommends as little as 10 pg to 10 ng of isolated RNA for 100,000 to 5 million paired-end reads per sample.

Why choose MERCURIUS™ BRB-seq over QuantSeq-Pool?

Comparative analyses between MERCURIUS™ BRB-seq and QuantSeq-Pool highlighted that MERCURIUS™ BRB-seq performed better than its competitor under the same conditions, with a lower cost per sample.

In representative experiments, MERCURIUS™ BRB-seq had a higher demultiplexing rate and number of uniquely mapped reads, and detected almost twice the number of differentially expressed genes compared to QuantSeq-Pool. The MERCURIUS™ BRB-seq kits therefore provide superior quality data.

To find out more about how MERCURIUS™ BRB-seq can help in your study, please contact us at info@alitheagenomics.com.