# RACE-Seq
## Requirements:
- Pre-processing of data
  - STAR
  - cutadapt
  - ConDeTri
  - BBMap
  - umi_tools
  - umicollapse
  - samtools
- Tails identification
  - get_softclipped_reads_from_sam.pl (from https://github.com/smaegol/LINE_1_RACE_seq_analysis)
  - seqtk
  - *R* libraries:
    - biomaRt
    - tidyr
    - stringr
    - dplyr
    - sqldf
    - stringi
    - optparse
- Visualization
  - *R* libraries
    - sqldf
    - ggplot2
    - dplyr
    - stringr
    - ggpubr
    - RColorBrewer
    - Ggridges

## Procedure
### I. Pre-processing
Raw reads are processed and map to the specific gene sequences. Pre-processing section include following steps: adapters trimming, removing of nucleotides from 3’ end of reads with low quality, amplicons filtering, reads containing full length of UMI+delimiter filtering, UMI extraction, mapping and deduplication.

**Script:** 01_data_preprocessing

**Files:** fastq files with raw reads, ref_sequence.fa, annotation.gtf
### II.	Tails identification
The identification of tails is done thanks to the presence of soft-clips in BAM/SAM files. Soft-clips are 5’ or 3’ parts of reads that do not map to the reference sequence, and information about them is retained in BAM/SAM files by STAR. This script utilizes information from the soft-clip presents in the 3' end of the read.

**Scripts:** Tail_analysis.sh, seq.R, poli_clip_all.R

**Files:** SAM files, annotation.gtf
### III.	Visualization
In this section, you can plot graphs showing the length distribution of tails categorized by their classes, the content of U nucleotides in AU tails or only U tails, and the U content in tails based on their length.
Scripts with X1KO in their names are prepared for data set included WT and XRN1-KO cell lines. Scripts with D2X1KO in their names are prepared for data set included WT, XRN1-KO, DCP2-KO and XRN1-DCP2-doubleKO cell lines.

**Scripts:** 03_visualization_L1_X1KO.Rmd, 03_visualization_GAPDH_X1KO.Rmd, 03_visualization_PABPC4_X1KO.Rmd, 03_visualization_functions_X1KO.R, 03_visualization_L1_D2X1KO.Rmd, 03_visualization_GAPDH_D2X1KO.Rmd, 03_visualization_functions_D2X1KO.R

**Files:** *.softclips.results.csv files, RACESeq_infos.csv