Fig. 2

RNA sequencing technologies and workflows of qPCR, microarray, Illumina NGS, PacBio and ONT (Oxford Nanopore Technology). A cDNA is synthesized by reverse transcription of extracted RNAs. Fluorescent signals are emitted and detected by the qPCR instruments during PCR. The figure shows the basic principle of SYBR green detection. The quantitative real-time PCR amplification plot is shown at the bottom. The number of PCR cycles is shown on the x-axis, and the fluorescence from the amplification reaction, which is proportional to the amount of amplified product in the tube, is shown on the y-axis. B After the extracted RNA targets are converted to cDNA /cRNA templates, they can hybridize with the cDNA probes on the chips. The cDNA / cRNA samples would carry signals and are labeled with fluorescent tags. When target cDNA / cRNA templates bind to the complementary oligonucleotide probes, the signals will be released. The signal intensities correspond to the abundance of cDNA / cRNA binds to the probes. Each dot observed in the bottom figure represents a cumulative hybridization reaction. Red color denotes higher expression levels of experimental groups while green color denotes higher expression levels of the control group. cDNA arrays typically involve two channels (two colors in the B), but single channel (one color) is also available [178]. C After cDNA library preparation, individual cDNA molecules are clustered on a flow cell. Illumina NGS detects the sequences by synthesis using fluorescent labelled nucleotides. In each small step of sequencing, the growing DNA strand will emit signals from one of the four fluorophores when the nucleotide has been incorporated (images are modified from [202]. The emission wavelength and intensity are used to identify the bases. D After PacBio SMRT-adaptor ligation, circularized cDNA molecule is formed. The individual molecules are loaded into a sequencing chip, where they bind to a polymerase immobilized at the bottom of a nanowell. As each of the fluorescently labelled nucleotides is incorporated into the growing strand, the fluorescent signals are emitted and detected by the PacBio instrument (images are modified from [202]. cDNA sequencing on the PacBio platform enables full-length sequencing from 5’ cap to the 3’ RNA cleavage site. (E) After library preparation, individual molecules attached with motor protein during adaptor ligation are loaded into a flow cell. The motor protein controls the translocation of the RNA strand through the nanopore, causing a change in current that is characteristic for the subsequent bases and will serve as the basis for basecalling. The figure at the bottom shows the corresponding electrical currents (in the pA-range) to nucleotides. The pros and cons of all platforms are listed in the Additional file 1. The numbers of 1, 2, 3 correspond to very good, good and fair performance of each platform. N.A. non-available