Περίληψη:
Background: Technological 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.
Results: The 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 KLKgene family members, but also
identified novel, previously uncharacterized ones.
Conclusions: In 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.
Συγγραφείς:
Adamopoulos, Panagiotis G.
Tsiakanikas, Panagiotis
Stolidi,
Irene
Scorilas, Andreas
