The cDNA products are amplified using a poly(T) adapter along with the first GSP primer that anneals upstream compared to GSP used in reverse transcription. Thereafter, a poly(A) tail is incorporated in cDNA products, using terminal deoxynucleotidyl transferase (TdT) and dATP. In homopolymeric tailing approaches a GSP primer is used to generate first-strand synthesis products. Initial attempts included the use of homopolymeric tailing or ligation anchored tailing. The successful identification of novel 5′ UTRs is of high significance, since these regions are firmly associated with mRNA stability, the subcellular localization and/or the translational efficiency of mRNAs. As a result, the major challenge of 5′ RACE compared to the 3′ RACE is the accurate incorporation of an adapter sequence in the 5′ end of the mRNAs that will lead to the unbiased characterization of 5′ UTRs. Īlthough similar approaches have also been employed for the characterization of the 5′ UTRs, 5′ RACE is often a more complicated and challenging procedure, since the 5′ ends of mRNAs lack any generic priming sites. This approach enables the identification of any unknown mRNA sequence located between this specific exon and the poly(A) tail. 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. In the first step, mRNAs are reversely transcribed into cDNA, using a reverse transcriptase and an oligo-dT adapter as primer. The 3′ RACE is a well-described 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. Since its establishment in 1988, RACE has emerged as the main strategy used to determine both the 5′ and/or the 3′ untranslated regions (UTRs) of any mRNA transcript, defining the transcription start point(s) as well as the poly(A) tail sites, accordingly. Rapid amplification of cDNA ends (RACE), also described as “one-sided” PCR or “anchored” PCR, is a molecular technique that enables the amplification of nucleic acid sequences from a messenger RNA (mRNA), between a specific internal region and either the 3′ or the 5′ end of the mRNA. 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. In this work we present an in-house developed 5′ RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. Our results confirmed the existence of multiple annotated 5′ UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. 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. 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. 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. The optimization of the described 5′ RACE-seq method was accomplished using the human BCL2L12 as control gene. 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. 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. However, 5′ ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome.
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