identificaiton and differential expression analysis of CHO cell long non-coding lncRNA

These scripts replicate the results of the following manuscript

Installation

Dependancies

RNASeq read QC, alignment and genome guided transcriptome assembly

TBC

get the ensembl CHOK1 genome and GTF

./scripts/prepare_genome.sh -v 94 -o reference_genome

make a STAR index for alignment

-a = genome fasta file -g = anntation file -p = processors

./scripts/make_star_index.sh -a reference_genome/ensembl_chok1_genome.fa -g reference_genome/ensembl_chok1_genome.gtf -p 32

create a list of sample sample_names

ls ../data/raw/ | sed -n 's/\.fastq.gz$//p' | cut -d_ -f1-2 | uniq > ../data/sample_names.txt

trim adapter sequences

cat ../data/sample_names.txt | while read sample; do
./scripts/trim_adapter.sh -s $sample  -i ../data/raw -o ../data/preprocessed/cutadapt&
done

quality trimming

cat ../data/sample_names.txt | while read sample; do
./scripts/trim_quality.sh -s $sample -i ../data/preprocessed/cutadapt -o../data/preprocessed
done

map to CHOK1 genome

cat ../data/sample_names.txt | while read sample; do
./scripts/star_mapping.sh -s $sample -i ../data/preprocessed/paired -g reference_gene/star_index -o mapped -p 32
done

string tie assembly

cat ../data/sample_names.txt | while read sample; do
./scripts/stringtie_star.sh -s $sample mapping -i mapped -g reference_genome/ensembl_chok1_genome.gtf -o stringtie_output -p 32
done

merge individual stringtie assemblies and compare to ENSEMBL annotation

./scripts/stringtie_merge.sh -t stringtie_output reference_genome/ensembl_chok1_genome.gtf

calculate TPM expression values

cat ../data/sample_names.txt | while read sample; do
./scripts/run_TPM.sh -p 32 -s $sample -g stringtie_output/stringtie_merged.gtf -o lncrna_annotation/TPM -b ../alt_splicing_analysis/mapping/
done

aggregate expression for each sample

./scripts/make_TPM_matrix.sh -s ../data/sample_names.txt -o lncrna_annotation/TPM/

Long non-coding RNA identification

The stringtie transcriptome assembly is used to predict lncRNAs using FEELNc

make a directory for the lncRNA annotation steps

mkdir -p lncrna_annotation

FEELNc analysis

The stringtie transcriptome assembly is used to predict lncRNAs using FEELNc

./scripts/run_FEELnc.sh -G reference_genome/ensembl_chok1_genome.gtf -g stringtie_output/stringtie_merged.gtf -f reference_genome/ensembl_chok1_genome.fa -o lncrna_annotation/FEELnc

Use transdecoder to create a cDNA fasta file identify the longest ORF for each candidate lncRNA

./scripts/run_Transdecoder.sh -g lncrna_annotation/FEELnc/candidate_lncRNA.nocodpot.gtf -f reference_genome/ensembl_chok1_genome.fa -o lncrna_annotation/TRANSDECODER

CPAT coding prediction for FEELNc candidate lncRNAs

./scripts/run_CPAT.sh -f lncrna_annotation/TRANSDECODER/candidate_lncRNA.nocodpot.cdna.fa -o lncrna_annotation/CPAT

CPC2 coding prediction for FEELnc candidate lncRNAs

./scripts/run_CPC2.sh -f lncrna_annotation/TRANSDECODER/candidate_lncRNA.nocodpot.cdna.fa -o lncrna_annotation/CPC2

Assess FEELnc candiate lncRNAs for the presence of protein domains

./scripts/run_HMMscan.sh -t 32 -e 1e-5 -p lncrna_annotation/TRANSDECODER/longest_orfs.pep -o lncrna_annotation/PFAM

Assess FEELnc candiate lncRNAs for the presence of proteins, miRNAs, and other non-coding RNAs (e.g. snoRNAs) using BLAST

./scripts/run_BLAST.sh  -t 32 -e 1e-5 -s lncrna_annotation/SWISSPROT -p lncrna_annotation/TRANSDECODER/longest_orfs.pep -m lncrna_annotation/MIRBASE -r lncrna_annotation/RFAM -n lncrna_annotation/TRANSDECODER/candidate_lncRNA.nocodpot.cdna.fa

Filter FEELNc output using additional protein potential calculators, PFAM search and BLAST against protein and RNA databases

Rscript R/filter_lncrna.R \
  "lncrna_annotation/FEELnc/candidate_lncRNA.nocodpot.gtf" \
  "lncrna_annotation/CPC2/CPC2.analysis.txt" \
  "lncrna_annotation/CPAT/CPAT.analysis.txt" \
  "lncrna_annotation/SWISSPROT/blastp.outfmt6" \
  "lncrna_annotation/MIRBASE/blastn.outfmt6" \
  "lncrna_annotation/PFAM/pfam_domain_transcripts" \
  "lncrna_annotation/RFAM/blastn.outfmt6" \
  "lncrna_annotation/TPM/transcript_tpm_all_samples.tsv" \
  "lncrna_annotation/FEELnc/lncRNA_classes.txt" \
  "stringtie_output/stringtie_merged.gtf" \
  "lncrna_annotation"

Differential expression analysis

cat ../data/sample_names.txt | while read sample; do
./scripts/htseq_count.sh -s $sample -m ../alt_splicing_analysis/mapping/ -g stringtie_output/stringtie_merged.gtf -o ./htseq_counts&
done

count for DESeq2

Rscript R/run_DEseq2.R \
  "htseq_counts" \
  "DESeq2_results" \
  "lncrna_annotation/lncRNA_filtering.rData"