README.md

Core for "De novo circular RNA represents a novel class of HCMV transcripts"

Human cytomegalovirus (HCMV) infects a large portion of the human population globally. Several HCMV-derived non-coding RNAs are involved in the regulation of viral gene expression and the virus lifecycle. Here, we report that circRNAs are a new class of HCMV transcripts. We bioinformatically predict 704 candidate circRNAs encoded by TB40/E strain and 230 encoded by the HAN strain. We also systematically compare circRNA features, including the breakpoint sequence consensus, strand preference, length distribution, and exon numbers between host genome-encoded circRNAs and viral circRNAs and show that the unique characteristics of viral circRNAs are correlated with their genome types. Furthermore, we experimentally confirm 324 back-splice junctions (BSJs) from three HCMV strain, Towne, TB40/E and Toledo and identify 4 representative HCMV circRNAs by RNase R treatment. Interestingly, we also show that HCMV contains alternative back-splicing circRNAs. We develop a new amplified FISH method that allows us to not only visualize circRNAs but also to quantify the number of circRNA molecules in the infected cells. The competitive endogenous RNA network analysis suggests that HCMV circRNAs play important roles in viral DNA synthesis via circRNA-miRNA-mRNA networks. Our findings highlight that circRNAs are an important component of the HCMV transcriptome that may contribute to viral replication and pathogenesis.

Requirements

Computer

Two Intel W-3175X CPUs with 128 GB memory running Ubuntu system (version 18.04)

Tools

• 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.ViReMa https://sourceforge.net/projects/virema/

Data preparation

Data collection

• 1.HCMV HAN strain infected HELF cells at 72 hours post-infection (PRJNA577553) https://www.ebi.ac.uk/ena/browser/view/PRJNA577553
• 2.HCMV TB40/E strain infected primary fibroblasts (HFF), endothelial cells (EC), and neural progenitors (NPCs) derived from embryonic stem cells at 48 hpi and 96 hpi (PRJNA299678)
• 3.KSHV BCBL1 strain infected B-cell lymphoma cells with RNase R-treatment (PRJNA483204)
• 4.EBV Akata strain infected B-cell lymphoma cells with RNase R-treatment (PRJNA479852)

Download RNA_seq data from ebi

for i in vol1/fastq/SRR102/087/SRR10277187/SRR10277187_1.fastq.gz vol1/fastq/SRR102/087/SRR10277187/SRR10277187_2.fastq.gz
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

for i in your_SRR*_id
do
fasterq-dump ${i} -3 -e 16 -p
echo ${i}
done
rm SRR*

Generating genome contained human and HCMV(HAN)

cat hg19.fa KJ426589.1.fasta > hg19_HAN.fa
cat hg19.gtf KJ426589.1.gtf > hg19_HAN.gtf

Build BWA index

bwa index hg19_HAN.fa

running CIRI2 and circ-full pipeline

Illustration of CIRI2-based identification of circRNAs

for i in SRR10277187
do
echo $i
mkdir ${i}_output
bwa mem -t 52 hg19_HAN.fa ${i}_1.fastq.gz ${i}_2.fastq.gz >${i}_output/${i}.sam
perl ~/CIRI2/CIRI_v2.0.6/CIRI2.pl -I ${i}_output/${i}.sam -O ${i}_output/${i}.ciri -F hg19_HAN.fa -A hg19_HAN.gtf -T 24
## Reconstructed HCMV circRNAs circ-full
perl ~/CIRI2/CIRI_AS/CIRI_AS_v1.2.pl -S ${i}_output/${i}.sam -C ${i}_output/${i}.ciri -F hg19_HAN.fa -A hg19_HAN.gtf -O ${i}_output/${i} -D yes
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar RO1 -1 ${i}_1.fastq.gz -2 ${i}_2.fastq.gz -o ${i}_output/${i}
bwa mem -t 52 hg19_HAN.fa ${i}_output/${i}_ro1.fq > ${i}_output/${i}_ro1.sam
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar RO2 -r hg19_HAN.fa -s ${i}_output/${i}_ro1.sam -l 300 -o ${i}_output/${i}RO2
java -jar ~/CIRI2/CIRI-full_v2.0/CIRI-full.jar Merge -c ${i}_output/${i}.ciri -as ${i}_output/${i}_jav.list -ro ${i}_output/${i}RO2_ro2_info.list -a hg19_HAN.gtf -r hg19_HAN.fa -o ${i}_output/${i}
unset DISPLAY
java -jar ~/CIRI2/CIRI_vis/CIRI-vis_v1.4.jar -i ${i}_output/${i}_merge_circRNA_detail.anno -l ${i}_output/${i}_library_length.list -r hg19_HAN.fa -min 1
echo ${i}_CircRNA_full_finished
done
  

running ViReMa pipeline

bowtie-build KJ426589.1.fasta KJ426589.1.fasta

for i in SRR10277187
do
trim_galore --phred33 --gzip --trim-n -o trim_galore_out_dir --paired ${i}_1.fastq.gz ${i}_2.fastq.gz
echo ${i}_trim_galore
cat trim_galore_out_dir/${i}_1_val_1.fq.gz trim_galore_out_dir/${i}_2_val_2.fq.gz > ${i}.fq.gz
python2 ViReMa.py VZV_HSV1.fasta trim_galore_out_dir/${i}.fq.gz ${i}.sam --Output_Dir ${i}_virema/ --Output_Tag ${i} -BED --p 50 --MicroInDel_Length 5 --Defuzz 0 -FuzzEntry
done

running statistics pipeline

mapping statistics

for i in SRR10277187
do
echo $i
samtools view -bS ${i}_output/${i}.sam > ${i}_output/${i}.bam
rm ${i}_output/${i}.sam
samtools sort ${i}_output/${i}.bam -o ${i}_output/${i}_sorted.bam -@ 42
qualimap bamqc -bam ${i}_output/${i}_sorted.bam -oc count.matrix -outdir ${i}_output/${i}_bamqc -outformat PDF:HTML --java-mem-size=50G
rm ${i}_output/${i}.bam
done

genome coverage statistics

for i in SRR10277187
do
samtools index -b ${i}_output/${i}_sort.bam ${i}_output/${i}_sort.bam.bai
samtools idxstats ${i}_output/${i}_sort.bam
samtools depth -a -m 0 ${i}_output/${i}_sort.bam > ${i}_output/${i}_coverage.txt
done

Citations

1. Yang S, Liu X, Wang M, Cao D, Jaijyan DK, Enescu N, Liu J, Wu S, Wang S, Sun W, Xiao L, Gu A, Li Y, Zhou H, Tyagi S, Wu J, Tang Q, Zhu H. Circular RNAs Represent a Novel Class of Human Cytomegalovirus Transcripts. Microbiol Spectr. 2022 May 23:e0110622. doi: 10.1128/spectrum.01106-22. Epub ahead of print. PMID: 35604147.
2. 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
3. Yuan Gao†, Jinfeng Wang† and Fangqing Zhao*. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biology (2015) 16:4.
4. Yuan Gao, Jinyang Zhang and Fangqing Zhao*. Circular RNA identification based on multiple seed matching. Briefings in Bioinformatics (2017) DOI: 10.1093/bib/bbx014.
5. Routh A, Johnson JE. Discovery of functional genomic motifs in viruses with ViReMa-a Virus Recombination Mapper-for analysis of next-generation sequencing data. Nucleic Acids Res. 2014 Jan;42(2):e11. doi: 10.1093/nar/gkt916. Epub 2013 Oct 16. PMID: 24137010; PMCID: PMC3902915.