A short question, can I use this tool for total RNA sequencing (RNA without rRNA)?

[hjsbio]

I am working on a project, which include RNA-seq data generated by total RNA library sequencing, with no specific filter for mRNA or enrichment for nascent RNA. Using IGV, my mapping bam files included a lot of mapped reads from nascent RNA. May I use PEPPRO in my project? There are a lot of Total RNA-sequencing available in open database, It will be a widely need for processing these data including nascent RNA.

[jpsmith5]

Hi @hjsbio, sorry for the delay getting back to you here. In general, I'd say not really. Many of the processing steps in the pipeline are really expecting the source to be from nascent technologies. You could specify custom adapters and turn off reverse complementation by setting the library as GRO-seq, but those are still just half-measures to treat it as a RNA-seq pipeline. While it will take RNA-seq data as a source and run successfully, it isn't designed for the typical RNA-seq pipeline. Particularly, downstream processes split the data into (at least for paired-end) just read1 and read2 and utilizes only the read1 information for signal track generation for example which is of more interest in nascent-seq processing. It would essentially be reporting RNA-seq as contaminants to the nascent. Let me know if that helps clear it up at all, or if I can help more.

[hjsbio]

Thanks Jason Smith, and sorry for the delay replying back to you. I will check the details of the tool and to see whether I can apply it to my data. my RNA-seq is strand-specific, I will quantify the expression level of one gene both including the exon and intron (e.g. the transcripts). Also, I could also only quantify the intron part of one gene, and this may help to estimate the contribution of nascent RNA to the total expression level of one gene. Also, I will check other analysis in this tool, and report my results.