A Snakemake-based pipeline to annotate novel lncRNA using existing annotation.
This pipeline uses various bioinformatics tools to annotate lncRNA. It requires reference genome(s) (optional HISAT index), sequencing reads, and reference annotation(s).
The workflow is mainly divided into two parts:
The first part of pipeline works with the following steps:
- uses HISAT2 to align all reads to the transcriptome,
- creates a new gene list with all known and novel genes.
- separates the novel genes
- uses CPAT and CPC to annotate novel genes with coding potential
- extracts non-coding genes
- identifies and removes transcripts with coding isoforms and less than 200 bp long genes
The second part of pipeline works with the following steps:
- uses StringTie to merge lncRNAs from different sample
- creates transcriptome using GFFread
- creates Salmon index for transcriptome
- quantify lncRNAs of each replicate of tissue/sample using salmon
- creates a TPM matrix for each sample and replicates
- filter lncRNAs with low expression value
A simple overview of pipeline is shown below:
| Part 1 (Snakefile) | Part 2 (Snakefile_quantification) |
|---|---|
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This pipeline uses conda environments. Snakemake will create conda environments during the first run of the pipeline. However, if running this pipeline on High-Performance Computing (HPC) it may not have an active internet connection. In that case, conda environment can be created using snakemake -s env_snakefile --useconda command from the node with an internet connection. And then the pipeline will be able to utilise existing environments.