3' mRNA-seq

3’ mRNA-seq is a quantitative, genome-wide transcriptomic technique based on the barcoding of the 3’ untranslated region (UTR) of mRNA molecules. Unlike standard bulk RNA-seq, where short sequencing reads are generated along the entire length of mRNA transcripts, only the 3’ end of polyadenylated RNAs are sequenced in 3’ mRNA-seq. This approach results in a need for fewer reads to quantify the expression of a gene and reduces the sequencing depth required per sample while providing robust and reliable transcriptome-wide read-outs of gene expression levels comparable to full-length RNA-seq methods.

Sample barcoding and the reduced per-sample sequencing depth also allow higher levels of sample multiplexing per experiment and lower the cost of transcriptome sequencing compared to full-length RNA-seq methods. These factors are crucial for large-scale, ultra-high-throughput gene expression studies or studies assessing differential gene expression between different experimental conditions or cell types (1 Alpern et al).

Some 3’ mRNA-seq technologies, like Bulk RNA Barcoding and Sequencing (BRB-seq) commercialized by Alithea Genomics further streamline the library preparation process by pooling up to 384 samples very early in the workflow for a cost per sample tantamount to profiling four individual genes using conventional qRT-PCR, in a workflow requiring less than two and a half hours hands-on time. An increasing number of 3’ mRNA-seq techniques also include unique molecular identifiers (UMIs) in sample barcodes to uniquely label each mRNA molecule and to distinguish between original mRNA transcripts and duplicates that result from PCR amplification.

History

The sample barcoding approach used in 3’ mRNA-seq was first established in the field of single-cell transcriptomics, where sample and mRNA barcoding allowed hundreds to thousands of single cells to be multiplexed in one experiment. Single-cell RNA profiling technologies like CEL-seq2, SCRB-seq, and STRT-seq also allowed the pooling of large sets of samples into one unique sequencing library at an early stage in the protocol.

Method

While numerous different 3’ mRNA-seq methods are available, their fundamental principles are generally similar.

Each method relies on an initial reverse transcription step in which mRNAs are labeled with sample barcodes. Reverse transcription can be performed with oligo dT primers, barcoded oligo dT primers, and template-switching oligos. In contrast, bulk RNA-seq library preparation methods like Illumina TruSeq mRNA Stranded kits use random priming of pre-fragmented RNA for reverse transcription to ensure reads are generated along the entire length of mRNA transcripts.

Second-strand synthesis is then performed in each method by DNA polymerase 1, nick translation or PCR, resulting in double-stranded complementary DNA (cDNA). This is followed by a process called tagmentation, in which double-stranded cDNA is fragmented and tagged using Tn5 transposase, which cleaves the cDNA and ligates adaptors for library amplification. Some methods use random primers for this stage.

Library indexing and PCR amplification then take place, resulting in libraries enriched for the 3’ untranslated region of mRNAs and suitable for short-read sequencing on Illumina or MGI sequencing instruments.

Advantages of 3’ mRNA-seq

Cost

3’ mRNA-seq methods are generally cheaper per sample than standard bulk RNA-seq methods. This is because of the lower sequencing depth required as only the 3’ end of mRNA molecules are sequenced instead of the whole length of entire transcripts. Read depths of between one million and five million reads are recommended in commercialized 3’ mRNA-seq protocols and are suitable for detecting the majority of highly expressed genes . For methods where samples are pooled early in the workflow, consumable use is also reduced. For instance, BRB-seq is up to 25 times cheaper than Illumina TruSeq stranded mRNA library preparations, with a cost equivalent to assessing four genes by RT-qPCR.

Sample throughput

3’ mRNA-seq methods have a higher sample throughput than standard RNA-seq approaches. Different options allow up to 384 samples to be barcoded and multiplexed at different stages in the library preparation protocol followed by short-read sequencing. 3’ mRNA-seq methods are performed in 96 or 384 well plates and are suitable for automated pipelines.  

Ease of data analysis

As only the 3’ end of mRNA molecules are sequenced, read counts do not need to be normalized for transcript length as in standard RNA-seq methods. Similarly, the data size per sample is lower than for standard RNA-seq due to the lower sequencing depth required. This simplifies data analysis with options available for rapid cloud-based analyses.

Suitable for degraded RNA

3’ mRNA-seq is suitable for high-quality and degraded RNA with RIN < 6. It is largely insensitive to RNA degradation because only the 3’ region of mRNA transcripts are prepared for sequencing.

Limitations of 3’ mRNA-seq

3’ mRNA-seq methods do not allow for the analysis of full-length transcripts, splice variants, fusion genes, or RNA editing.