Core for "SARS-CoV-2, SARS-CoV and MERS-CoV encode circular RNAs of spliceosome-independent origin"

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using bioinformatical and experimental approaches. We predicted 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and experimentally identified 75 SARS-CoV-2 circRNAs.

Requirements

• 1.Bash (Ubuntu, version 18.04)
• 2.Perl https://www.perl.org
• 3.Java https://javadl.oracle.com
• 3.BWA ttp://bio-bwa.sourceforge.net
• 4.CIRI2 https://sourceforge.net/projects/ciri/files/CIRI2/
• 5.SAMtools http://www.htslib.org/
• 6.sratoolkit https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software
• 7.aspera https://www.ibm.com/products/aspera
• 8.Qualimap http://qualimap.conesalab.org/

Preparing Data

Download RNA_seq data (SARS-CoV-2-infected Vero E6 cells at 24 hours post-infection ,PRJNA644588) from ebi

for i in ftp.sra.ebi.ac.uk/vol1/srr/SRR121/098/SRR12164498 ftp.sra.ebi.ac.uk/vol1/srr/SRR121/099/SRR12164499 ftp.sra.ebi.ac.uk/vol1/srr/SRR121/000/SRR12164500
do
echo $i
ascp -QT -l 300m -P33001 \
-i ~/miniconda2/envs/rna/etc/asperaweb_id_dsa.openssh \
era-fasp@fasp.sra.ebi.ac.uk:/${i} .
done

Multi-threading to speed up the extraction of fastq from SRA-accessions

fasterq-dump --split-3 SRR* -e 16 -p

Pooling all samples together

cat SRR12164498_1.fastq SRR12164499_1.fastq SRR12164500_1.fastq > SARS_CoV_2_Vero_E6_24h_1.fastq
cat SRR12164498_2.fastq SRR12164499_2.fastq SRR12164500_2.fastq > SARS_CoV_2_Vero_E6_24h_2.fastq

Remove all SRR files

rm SRR*

Generating genome contained African green monkey and SARS-CoV-2

cat ChlSab1.1.101.fa NC_045512.2.fasta > ChlSab1.1.101_NC_045512.2.fa
cat ChlSab1.1.101.gtf NC_045512.2.gtf > ChlSab1.1.101_NC_045512.2.gtf

Build BWA index

bwa index ChlSab1.1.101_NC_045512.2.fa

Running CIRI2 and circ-full pipeline

Illustration of CIRI2-based identification of circRNAs

for i in SARS_CoV_2_Vero_E6_24h
do
echo $i
mkdir ${i}_ChlSab1.1.101_SARS_CoV_2_output
bwa mem -t 52 ChlSab1.1.101_NC_045512.2.fa ${i}_1.fastq ${i}_2.fastq >${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.sam
perl ~/CIRI2/CIRI_v2.0.6/CIRI2.pl -I ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.sam -O ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.ciri -F ChlSab1.1.101_NC_045512.2.fa -A ChlSab1.1.101_NC_045512.2.gtf -T 24
## Reconstructed SARS-CoV-2 circRNAs circ-full
perl ~/CIRI2/CIRI_AS/CIRI_AS_v1.2.pl -S ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.sam -C ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.ciri -F ChlSab1.1.101_NC_045512.2.fa -A ChlSab1.1.101_NC_045512.2.gtf -O ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i} -D yes
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar RO1 -1 ${i}_1.fastq -2 ${i}_2.fastq -o ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}
bwa mem -t 52 ChlSab1.1.101_NC_045512.2.fa ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_ro1.fq > ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_ro1.sam
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar RO2 -r ChlSab1.1.101_NC_045512.2.fa -s ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_ro1.sam -l 300 -o ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}RO2
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar Merge -c ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.ciri -as ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_jav.list -ro ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}RO2_ro2_info.list -a ChlSab1.1.101_NC_045512.2.gtf -r ChlSab1.1.101_NC_045512.2.fa -o ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}
unset DISPLAY
java -jar ~/CIRI2/CIRI_vis/CIRI-vis_v1.4.jar -i ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_merge_circRNA_detail.anno -l ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_library_length.list -r ChlSab1.1.101_NC_045512.2.fa -min 1
done
  

Running mapping statistics pipeline

for i in SARS_CoV_2_Vero_E6_24h
do
echo $i
samtools view -bS ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.sam > ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.bam
rm ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.sam
samtools sort ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.bam -o ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_sorted.bam -@ 42
qualimap bamqc -bam ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_sorted.bam -oc count.matrix -outdir ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}_bamqc -outformat PDF:HTML --java-mem-size=50G
rm ${i}_ChlSab1.1.101_SARS_CoV_2_output/${i}.bam
done

Citations

1. Yang S, Zhou H, Liu M, et al. SARSCoV‐2, SARS‐CoV, and MERS‐CoV encode circular RNAs of spliceosome‐independent origin. J Med Virol. 2022;1‐20. doi:10.1002/jmv.27734
2. Yuan Gao†, Jinfeng Wang† and Fangqing Zhao*. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biology (2015) 16:4.
3. Yuan Gao, Jinyang Zhang and Fangqing Zhao*. Circular RNA identification based on multiple seed matching. Briefings in Bioinformatics (2017) DOI: 10.1093/bib/bbx014.