"genomes/hg38/" # reference genomes (i.e. genome sequence and annotation)
"shared_scripts/" # shared scripts by Lu Lab
~/github # I sync my own scripts to github
~/proj_exRNA/
|-- RNA_index # rerference transcriptomes (fasta and index)
|-- sample*_name # ...
|-- sample2_name # different samples
`-- sample1_name
|-- fastq # raw data: fastq files
|-- fastqc # QC of fastq
|-- trim # trimmed fastq (e.g. 3' adaptor cutted)
|-- mapped # mapped data: SAM/BAM files
`-- exp # expression of each gene/ncRNA
Inputs
File format
Information contained in file
File description
Notes
bam
alignments
Produced by mapping reads to the transcriptome.
Reads are trimmed using a proprietary version of cutAdapt. We map to transcriptome for a better sensitivity (see details in protocol and example).
Outputs
File format
Information contained in file
File description
Notes
bigWig
signal
Normalized RNA-seq signal
Signals are generated for transcriptome both the plus and minus strands and for unique reads and unique+multimapping reads.
tsv
gene (ncRNA) quantifications
Non-normalized counts.
Running Scripts
Software/Tools
RSEM
homer
HTseq
FeatureCounts
Example of single case
# genome seuqneces and annotaions
ln -s /BioII/lulab_b/shared/genomes ~/genomes
export hg38=~/genomes/human_hg38/sequence/GRCh38.p12.genome.fa
export gtf=~/genomes/human_hg38/gtf
# working space
cd ~/proj_exRNA
# first convert relative positions to genomic coordinates
# then convert bam to bigWig, so that we can view it in genome browser
#####################################################################
#0.1 convert the relative positions on RNAs to genomic coordinates
#####################################################################
rsem-tbam2gbam miRNA.indexDir/ NC_1.miRNA.sam NC_1.miRNA.rsem.bam
#####################################################################
#0.2 two ways to convert bam/sam to bedGraph/bigwig for visulaization
#####################################################################
#0.2a Using homer
#Make tag directories for each sample (e.g. NC_1)
#works with sam or bam (samtools must be installed for bam)
makeTagDirectory NC_1.miRNA.tagsDir/ NC_1.miRNA.sorted.bam
## Add "-strand separate" for strand-specific sequencing
makeUCSCfile NC_1.miRNA.tagsDir/ -fragLength given -o auto
# (repeat for other tag directories)
