So far most of the reported full length transcriptome articles adopt the mode of "3+2". “3” refers to the third generation sequencing that detects more accurate structural variation. For the species with reference genome, it can accurately detect alternative splicing, fusion genes, and predict new genes. “2” refers to the second generation sequencing that achieve more accurate quantification than the third generation sequencing. For the species without reference genome, “3+2” sequencing strategy can provide accurate reference sequences, which is helpful for subsequent differential expression analysis. In addition, the "3+2" sequencing strategy can use the data of the second generation sequencing to correct the third generation sequencing data.

The full-length transcriptome sequencing is based on the PacBio sequencing platform, which can directly obtain the complete transcripts containing 5 ', 3'UTR, polyA tail. It overcomes the limitations of short assembly and incomplete information of transcripts of the species without reference genome.

Regarding to PacBio Sequel platform, it is recommended to have 40G sequencing data amount. If low expression genes are interested, then 60G even more data amount are suggested.

Both species with / without reference genomes are suitable.

It mainly depends on the research purpose. In general, if you want to get more comprehensive transcripts of a species, it is recommended to collect different tissue samples of this species to extract RNA, mixed RNA to get as comprehensive transcripts as possible. For example, roots, stems, leaves, flowers, fruits of plants; liver, kidney, intestine, skin, and other parts of animals are recommended to collect. If full-length transcriptome sequencing is for a certain tissue part, then different development stages of the tissue can be sampled.