Full LncRNA data analysis workflow from sample.fastq to IsoMat.txt
NOTE: Internal use only, do not publish this code without removing non-original files from /lib
1. Download this repository
2. Place your data in the project folder
Each replicate should be placed in its own folder, and the two sample names should have the format [replicate name]_R[1\2].fastq
3. Download genome files
Three files are required: [genome].fa, [genome].[ensemble].gtf, and [genome].[ensemble].gff3.
Place these files within the \lib\Genome folder.
4. Download Slncky annotation files
Place these within \lib\annotations.
5. Rename parameters within the snakemake file
GENOME_VERSION - set to your genome version (what you named the files in step 3).
ENSEMBL_VERSION - set to your ensemble version (what you named the files in step 3).
SLNCKY_ANNOTATIONS - set to your genome version (hg38 for human, mm10 for mouse)
6. Run snakemake --use-conda from within the project folder in a command line
7. Check replicate folders and output folder for results
Any steps pertaining to an individual replicate will be in that replicate's folder. All combined steps will be within \output.
\output\logs will give run information for each step and also any errors that occured.
- Some code within the snakemake file was adapted from previous work by Anthony Cesnik, credit is also denoted within the file.
GTFSharp: Cesnik, A. J.; Yang, B.; Truong, A.; Spiniello, M.; Steinbrink, M.; Shortreed, M. R.; Frey, B. L.; Jarrard, D. F.; Smith, L. M. “Long Noncoding RNAs AC009014.3 and Newly Discovered XPLAID Differentiate Aggressive and Indolent Prostate Cancers.” Translational Oncology, 2018, 11, 808–814.RSEM: Li B and Dewey CN (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12 (1), 323.STAR: Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, and Gingeras TR (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1), 15–21.stringtie: Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT & Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads Nature Biotechnology 2015, doi:10.1038/nbt.3122.slncky: Chen J, Shishkin AA, Zhu X, Kadri S, Maza I, Guttman M, Hanna JH, Regev A, and Garber M (2016). Evolutionary analysis across mammals reveals distinct classes of long non-coding RNAs. Genome Biol 17(1), 19.