RNAseq_lncRNA

Data

The data is composed of 48 fastq.gz files separeted in 4 subline. Each subline is composed of 12 files. The files are in the repository ../Project1/fastq

Merge the fastq.gz

Run fastq_merged2.sh which merge the two lanes of each fastqt.gz. The repository is /fastq_merged

Number of reads

Run the counting_reads.sh will gives the output reads_count.txt which display the number of reads in each fastq.gz files

FastQC and MultiQC

Run fastqc.sh. Input are the fastq_merged files. Output fastqc.zip and fastqc.html files. Run multiqc.sh. Input are fastqc.html files and the ouput is a multiqc.html

Read mapping

HISAT2

Create a index for hisat2, run hisat_index.sh which use as reference annotation, GRCh38.p13.genome.fa.gz. Return 10 files Run hisat2_run.sh. Input fastq_merged files. Output bam files for each sbuline replicates.

Samtools

Merge the bam file of each replicates in one global bam file for eacn subline: Run samtools_merge.sh. Input bam file. Ouput three bam files

Assembly

Stringtie

Run sringtie.sh. Input bam files, output gtf file for each subline. Use as annotation reference, gencode.v35.annotation.gtf Run stringtie_merge.sh to merged the gtf files in one gtf file called stringtie_merged.gtf (meta assembly)

Quantification

Run kallisto_index.sh which will create hg38.transcript.idx file through GRCh38.p13.genome.fa and stringtie_merged.gtf which will be use for kallisto Run kallisto.sh. Input, fastq merged, Output abudance.h5, abundance.tsv and run_info.json for each subline replicates Analyse the numbers of transcripts, novel transcript and tpm running the file analysis_kallisto.sh

Differential_expression

Run sleuth.sh which will run sleuth.R. sleuth.R input kallisto outputs and return a sleuth_table.txt and a sleuth significante_txt locallised in results/Rfolder. Run sleuth_gene.R through sleuth.sh to add the gene names of the tables.

Inegrative analysis

PolyA, Cage and Intergenic.

Run polyA_cage_intergenic.sh. For cage: Input, cage_peaks_hg38 and transcript_file.gtf. Output, tss_transcript.txt For polyA: Input, transcript_file.gtf, atlas.clusters.2.0.GRCh38.96.bed. Output, transcript_polyA.txt For intergenic: Input, transcript_file.gtf , gencode.v35.annotation.gtf. Output, transcript_intergenic.txt

CPAT

Run cpat.sh

Final table generation

Run final_table.R and FFinal_script.R Outputs: table_under_2c.csv, table_under_4c.csv, table_under_8c.csv, table_over_2c.csv table_over_4c.csv, table_over_8c.csv and lncRNAs_literature.csv