Novel Method of Full-Length RNA-seq That Expands the Identification of Non-Polyadenylated RNAs Using Nanopore Sequencing

The occurrence of diseases displayed transcriptome alteration,
including both coding and non-coding transcripts. The third-generation
sequencing (TGS) technologies allow for intensive and comprehensive
research of the transcriptome. However, the present standard TGS RNA
sequencing method is unable to detect many of the non-polyadenylated
[non-poly(A)] RNAs. To obtain more complete transcriptome information,
we presented a new comprehensive sequencing approach by performing
conventional poly(A) RNA-sequencing combined with the sequencing of
non-poly(A) RNA fraction which was tailed by poly(U) on HepG2 and
HL-7702 cell lines, enabling the detection of multiple categories
of non-poly(A) RNAs excluded by the existing standard approach. Moreover,
the length distribution of the full-splice match transcripts was longer
than that assembled by short-reads, which contributed to characterizing
alternative splicing events and provided a comprehensive portrait
of transcriptional complexity. Besides the detection of genes with
differential expression patterns in the poly(A) library between HepG2
and HL-7702, we also found a cancer-related non-coding gene in the
poly(U) data, that is, growth arrest special 5 (GAS5). Collectively,
our results suggested that the novel method effectively captured both
poly(A) and non-poly(A) transcripts in the tested cell lines and allowed
a deeper exploration of the transcriptome.