Abstract
BackgroundTechnological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5′ ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5′ rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5′ ends of cDNAs. In the current study, we developed a 5′ RACE-seq method by coupling a custom template-switching and 5′ RACE assay with targeted nanopore sequencing, to accurately unveil 5′ termini of mRNA targets.ResultsThe optimization of the described 5′ RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5′ RACE. Collectively, our results support the existence of two distinct 5′ termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5′ RACE-seq methodology to investigate the 5′ UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5′ UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones.ConclusionsIn this work we present an in-house developed 5′ RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5′ UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.
Highlights
Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome
A nested 5′ rapid amplification of cDNA ends (RACE) assay was employed targeting the 5′ untranslated regions (UTRs) of the BCL2L12 gene, using two universal primers designed to anneal at the Template-switching oligo (TSO) sequence along with two gene-specific primer (GSP) (Supplementary Table 1)
The electrophoresis results clearly demonstrate that a DNA/RNA hybrid TSO is superior in terms of overall performance than a TSO composed of typical DNA nucleotides, which is in accordance with findings of previous studies [21]
Summary
Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. The 3′ RACE is a welldescribed and optimized methodology, which exploits the natural poly(A) tail of mRNAs as a generic priming site for PCR amplification and takes place in two distinct steps. The second step involves a PCR amplification using a gene-specific primer (GSP) that anneals to a region of a known exon sequence and a universal primer that is designed to anneal to the adapter sequence that was used in the previous step of the reverse transcription. This approach enables the identification of any unknown mRNA sequence located between this specific exon and the poly(A) tail [4]
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