seekCRIT

seek for circular RNA in transcriptome (identifies deferentially expressed circRNAs between two samples)

Version: v1.0.0.b

Last Modified: 2019-04-25

Authors: Bioinformatics Lab, University of Louisville, Kentucky Biomedical Research Infrastructure Network (KBRIN)

A schematic flow shows the pipeline

Different Junction Counts (linear junction counts and circular junction counts) used in the circular RNA expression level estimation.

Percent Back-spliced In (PBI) calculation (circular junction counts with respect to all junction counts)

Example PBI calculation for a differentially expressed circular RNA.

circular RNA derived from exon 2 of Cdp gene, Rat, Hippocampus, Somata and Neuropil. This circular RNA is highly expressed in Neuropil (31.9%) than Somata (11.9%).

Prerequisites

Software / Package

• STAR Aligner: v2.5.2b

Others

• pysam >=0.9.1.4

• numpy >=1.11.2

• scipy

• fisher (Fisher’s exact test)

• mne (FDR calculation)

Installation

1 Download seekCRIT

git clone https://github.com/UofLBioinformatics/seekCRIT.git

cd seekCRIT

2 Install required packages (Some prerequisite packages might require admin access rights. Please contact your system admin to install such packages.)

pip3 install -r Prerequisites.txt

3 Install seekCRIT

python setup.py install

4 testing seekCRIT with testrun.sh

In order to run testrun.sh:

• Dowload genome sequence in FASTA format for Rattus_norvegicus genome ( It can be downloaded from UCSC Genome Browser)
• Dowload gtf annotation for Rattus_norvegicus genome.
• Run testrun.sh to test the installation and the dependencies of seekCRIT and also it will test the installation with CRTL12.fastq and IR12.fastq by specifying the path for FASTA and gtf files:

  ./testrun.sh gtf/Rattus_norvegicus.Ensembl.rn6.r84.gtf fasta/rn6.fa

Usage

usage: seekCRIT.py [-h] -s1 S1 -s2 S2 -gtf GTF -o OUTDIR -t {SE,PE} 
               --genomeIndex GENOMEINDEX -fa FASTA -ref REFSEQ
               [--threadNumber numThreads]
               [--aligner aligner]
               [--deltaPSI DELTAPSI] [--highConfidence HIGHCONFIDENCE]
               [--libType {fr-unstranded,fr-firststrand,fr-secondstrand}]
               [--keepTemp {Y,N}]

Identifying and Characterizing Differentially Spliced circular RNAs between
two samples

Required arguments:
=================== 
  -s1 S1, --sample1 S1  fastq files for sample_1. Replicates are separated by
                        comma. Paired-end reads are separated by colon.
                        e.g.,s1-1.fastq,s1-2.fastq for single-end read. s1-1.R
                        1.fastq:s1-1.R2.fastq,s1-2.R1.fastq:s1-2.R2.fastq for
                        single-end read

  -s2 S2, --sample2 S2  fastq files for sample_2. Replicates are separated by
                        comma. Paired-end reads are separated by colon.
                        e.g.,s2-1.fastq,s2-2.fastq for single-end read. s2-1.R
                        1.fastq:s2-1.R2.fastq,s2-2.R1.fastq:s2-2.R2.fastq for
                        single-end read

  -gtf GTF, --gtf GTF   The gtf annotation file. e.g., hg38.gtf

  -o OUTDIR, --output OUTDIR
                        Output directory

  -t {SE,PE}, --readType {SE,PE}
                        Read type. SE for Single-end read, PE for Paired-end read

  --genomeIndex GENOMEINDEX
                        Genome indexes for the aligner

  -fa FASTA, --fasta FASTA
                        Genome sequence. e.g., hg38.fa

  -ref REFSEQ, --refseq REFSEQ
                        Transcriptome in refseq format. e.g., hg38.ref.txt

 optional arguments:
====================

   -h, --help            show this help message and exit
   --threadNumber numberOfThreadsk
                        Number of threads for multi-threading feature [default = 4]
  --aligner aligner     aligner to use(for now it supports only STAR but we are working on it to support more aligners)

  --deltaPSI DELTAPSI   Delta PSI cutoff. i.e., significant event must show
                        bigger deltaPSI than this cutoff [default = 0.05]

  --highConfidence HIGHCONFIDENCE
                        Minimum number of circular junction counts required [default = 1]

  --libType  {fr-unstranded,fr-firststrand,fr-secondstrand}
                        library type used by Tophat aligner [default ='fr-unstranded']

  --keepTemp {Y,N}      Keep temp files or not  [default='Y']

Example

Paired-end reads

python3 seekCRIT.py -o PEtest -t PE -fa fa/hg19.fa -ref ref/hg19.ref.txt --genomeIndex /media/bio/data/STARIndex/hg19 -s1 testData/231ESRP.25K.rep-1.R1.fastq:testData/231ESRP.25K.rep-1.R2.fastq,testData/231ESRP.25K.rep-2.R1.fastq:testData/231ESRP.25K.rep-2.R2.fastq -s2 testData/231EV.25K.rep-1.R1.fastq:testData/231EV.25K.rep-1.R2.fastq,testData/231EV.25K.rep-2.R1.fastq:testData/231EV.25K.rep-2.R2.fastq -gtf testData/test.gtf --threadNumber 12 

Single-end reads

python3 seekCRIT.py -o SEtest -t SE -fa fa/hg19.fa -ref ref/hg19.ref.txt --genomeIndex /media/bio/data/STARIndex/hg19 -s1 testData/231ESRP.25K.rep-1.R1.fastq,testData/231ESRP.25K.rep-1.R2.fastq,testData/231ESRP.25K.rep-2.R1.fastq,testData/231ESRP.25K.rep-2.R2.fastq -s2 testData/231EV.25K.rep-1.R1.fastq,testData/231EV.25K.rep-1.R2.fastq,testData/231EV.25K.rep-2.R1.fastq,testData/231EV.25K.rep-2.R2.fastq -gtf testData/test.gtf --threadNumber 12 

Note

• Transcriptome should be in refseq format below (see more details in the example ):

• It is not obligatory to provide REFSEQ file, we made script (GTFtoREFSEQ) to convert from gtf to refseq that is used in the main code if no refseq file is provided.

Output

See details in the example file

To calculate the significane of differentially expressed circular RNAs, use the criteria:

• At least 5% changes in percent back-spliced in (PBI) or |deltaPBI|>=5%

• FDR < 0.05

License

Copyright (C) 2017 . See the LICENSE file for license rights and limitations (MIT).