Analysis of DRS data from chikungunya virus

Scripts used in the analysis of Direct RNA nanopore sequencing (DRS) data included in article: Baquero-Perez et al., N6-methyladenosine modification is not a general trait of viral RNA genomes

Basecalling and demultiplexing:

The module mop_preprocess module from Master of Pores2 pipeline was used to perform basecalling, with guppy (version 3.1.5, model rna_r9.4.1_70bps_hac), and demultiplexing - when needed - with Deeplexicon (version 1.2.0). Then, it also mapped the basecalled reads to the suitable viral reference (for chikungunya: ref/Chikungunya_genome.fa and for adenovirus: ref/Ad5_transcriptome.fa) with miniamap2 (version 2.17) with unspliced parameters (-uf -k14 -ax map-ont). Moreover, same parameters were used to align reads to the to the human transcriptome from Ensembl, based on assembly GRCh38 (ref/HomoSapiens_GRCh38_transcriptome.fa.gz). To execute this module, please add these parameters into both params.config and tools_opt.tsv files and run the command below:

nextflow run mop_preprocess.nf -with-singularity -bg > log.txt

Extracting full-length reads::

• Generate bed file from bam file to know the alignment start and end for each read:

bedtools bamtobed -i CHK-HEK-Rep1.bam > CHK-HEK-Rep1.bam.bed

• Extracting reads' ID from full length reads:

#Subgenomic full-length reads: 
awk '{if ($1=="chikungunya" && $2<=8067 && $2>=7567 && $3>=11700) {print $4}}' CHK-HEK-Rep1.bam.bed > SubgenomicReads_CHK-HEK-Rep1.txt
 
#Genomic full-length reads: 
awk '{if ($1=="chikungunya" && $3-$2>=4517) {print $4}}' CHK-HEK-Rep1.bam.bed > GenomicReads_CHK-HEK-Rep1.txt

• Extracting basecalled fast5, fastq and alignments from full-length reads:

To extract basecalled fast5, fastq and alignments from full-length reads from specific samples, please use the script and the code below:

#Usage: 
bash ./scripts/Full_Length/FullLength_Basecalled_Fastq_Bam.sh /path/input/sample output_folder full-length_readsIDs.txt

#Examples:
bash ./scripts/Full_Length/FullLength_Basecalled_Fastq_Bam.sh CHK-HEK-Rep1 CHK-HEK-Rep1_SubgenomicFullLength SubgenomicReads_CHK-HEK-Rep1.txt 

Running RNA modification detection softwares:

EpiNano, Nanopolish, Tombo and Nanocompore were run by the module mop_mod module from Master of Pores2 with the default options for both EpiNano and Nanopolish whereas Tombo was run with additional options --percent-to-filter 80 and --multiprocess-region-size 50. Finally, Nanocompore was run with --downsample_high_coverage 5000. To execute this module, please add these parameters into tools_opt.tsv file and run the command below:

nextflow run mop_mod.nf -with-singularity -bg > log.txt

Running NanoConsensus:

NanoConsensus was run by the module mop_consensus from Master of Pores2 with both default parameters and relaxed ones (--MZS 3.75 and --NC 4).

nextflow run mop_consensus.nf -with-singularity -bg > log.txt

Dependencies

• Singularity (version 3.2.1)
• Nextflow (version 23.04.1)
• Bedtools (version 2.29.2)

Citation

Baquero-Perez B*, Yonchez ID*, Delgado-Tejedor A*, Medina R,Puig-Torrents M, Sudbery I, Begik O, Wilson SA, Novoa EM, Diez J. N6-methyladenosine modification is not a general trait of viral RNA genomes. Nature Communications 2024.