CDCscreen

A computational pipeline for Cas13d-mediated circRNA screen (CDCscreen) to identify negatively selected functional circular RNAs.

---

Schema

Maintainer: Wei Xue (xuewei@picb.ac.cn)

---

Installation requirements [mandatory]

• Software
  • perl (version 5.26.2)
  • bowtie (version 1.1.2)
  • cutadapt (version 1.18)
  • samtools (version: 1.9)
  • MAGeCK (version 0.5.9.2)
  • R (version 3.5.1)

Data requirements [mandatory]

• Cas13d BSJ-gRNA reference sequences
  • ref_gRNA_seq.fa
• Expression (FPBcirc) of circRNAs in examined cells
  • FPBcirc.txt (FPBcirc of circRNA is calculated with CLEAR)
• Raw FASTQ files (Paired-End reads, 2 biology replicates of control [Day 1] and treatment [Day 30], full path of raw data is recommended)
  • D1_rep1_R1.fq and D1_rep1_R2.fq (Day 1 of biology replicate 1)
  • D1_rep2_R1.fq and D1_rep2_R2.fq (Day 1 of biology replicate 2)
  • D30_rep1_R1.fq and D30_rep1_R2.fq (Day 30 of biology replicate 1)
  • D30_rep2_R1.fq and D30_rep2_R2.fq (Day 30 of biology replicate 2)

Installation

git clone https://github.com/YangLab/CDCscreen.git

Usage

'run_CDCscreen_2_reps.sh' is used for 2 biology replicates

sh run_CDCscreen_2_reps.sh ref_gRNA_seq.fa FPBcirc.txt D1_rep1_R1.fq D1_rep1_R2.fq D1_rep2_R1.fq D1_rep2_R2.fq D30_rep1_R1.fq D30_rep1_R2.fq D30_rep2_R1.fq D30_rep2_R2.fq

'run_CDCscreen_3_reps.sh' is used for 3 biology replicates

sh run_CDCscreen_3_reps.sh ref_gRNA_seq.fa FPBcirc.txt D1_rep1_R1.fq D1_rep1_R2.fq D1_rep2_R1.fq D1_rep2_R2.fq D1_rep3_R1.fq D1_rep3_R2.fq D30_rep1_R1.fq D30_rep1_R2.fq D30_rep2_R1.fq D30_rep2_R2.fq D30_rep3_R1.fq D30_rep3_R2.fq

Output

• CDCscreen_circRNA.txt is the result of CDCscreen pipeline.

---

Example: 'run_CDCscreen_2_reps.sh' is used for 2 biology replicates

1. Input data [mandatory]

• Test Cas13d BSJ-gRNA reference sequences file is in directory of 'test_data'

  • test_data/ref_gRNA_seq.fa

• Test expression (FPBcirc) of circRNAs of 293FT cells is in directory of 'test_data'. Ribo— RNA-seq raw data are downloaded from NCBI GEO dataset (GSE149691) and National Omics Data Encyclopedia (OEP000888).

  • test_data/FPBcirc_293FT.txt

• Test raw data (2 biology replicates of D1 and D30 in 293FT cells) are downloaded from NCBI GEO dataset (GSE149692) and National Omics Data Encyclopedia (OEP000889). Full path of raw data is recommended.

  • D1_rep1_R1.fq (FASTQ file of day 1 biology replicate 1 R1)
  • D1_rep1_R2.fq (FASTQ file of day 1 biology replicate 1 R2)
  • D1_rep2_R1.fq (FASTQ file of day 1 biology replicate 2 R1)
  • D1_rep2_R2.fq (FASTQ file of day 1 biology replicate 2 R2)
  • D30_rep1_R1.fq (FASTQ file of day 30 biology replicate 1 R1)
  • D30_rep1_R2.fq (FASTQ file of day 30 biology replicate 1 R2)
  • D30_rep2_R1.fq (FASTQ file of day 30 biology replicate 2 R1)
  • D30_rep2_R2.fq (FASTQ file of day 30 biology replicate 2 R2)

2. Usage

sh run_CDCscreen_2_reps.sh test_data/ref_gRNA_seq.fa test_data/FPBcirc_293FT.txt D1_rep1_R1.fq D1_rep1_R2.fq D1_rep2_R1.fq D1_rep2_R2.fq D30_rep1_R1.fq D30_rep1_R2.fq D30_rep2_R1.fq D30_rep2_R2.fq

3. Output

• test_data/CDCscreen_circRNA_293FT.txt is output of CDCscreen pipeline in 293FT cells.

Field	Description
chrom	Chromosome
start	Start of circRNA
end	End of circRNA
geneName	Gene symbol of circRNA
FPBcirc	Expression of circRNA
CDCscreen	CDCscreen score of circRNA

---

Citation

Siqi Li#, Xiang Li#, Wei Xue#, Lin Zhang#, Liang-Zhong Yang, Shi-Meng Cao, Yun-Ni Lei, Chu-Xiao Liu, Si-Kun Guo, Lin Shan, Man Wu, Xiao Tao, Jia-Lin Zhang, Xiang Gao, Jun Zhang, Jia Wei, Jinsong Li*, Li Yang*, Ling-Ling Chen*. Screening for functional circular RNAs using the CRISPR-Cas13 system. Nat Methods, 2020.

---

License

Copyright (C) 2020 YangLab. Licensed GPLv3 for open source use or contact YangLab (yanglab@@picb.ac.cn) for commercial use.