FLEP-seq (full-length elongating and polyadenylated RNA sequencing) allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally.
This pipeline includes basecalling and preprocessing of the FLEP-seq data.
## Software and package requirements
* Minimap2 (https://github.com/lh3/minimap2)
* SAMtools (http://www.htslib.org/)
* BLAST+ (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) (require for Nanopore data analysis)
| *.read.splicing_kinetics.pdf | splicing kinetics | Generated by `plot_intron_splicing_kinetics.R` |
## Step by step workflow
1. Nanopore basecalling
You can use MinKNOW to perform real-time basecalling while sequencing, or use the GPU version of Guppy to speed up basecalling after sequencing. Both MinKNOW and Guppy are available via Nanopore community site (https://community.nanoporetech.com). Command-line for running Guppy basecalling is as follow:
CAUTION: For the organisms with short introns, such as Arabidopsis, it might be better to use the parameter “-G” to set the max intron length, for example, “-G 12000”. You also can set “-t number_of_threads” to use more threads to speed up.
Long, Y., Jia, J., Mo, W. et al. FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing. Nat Protoc 16, 4355–4381 (2021). https://doi.org/10.1038/s41596-021-00581-7
Jia, J., Long, Y., Zhang, H. et al. Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants. Nat. Plants 6, 780–788 (2020). https://doi.org/10.1038/s41477-020-0688-1
