plot_ORFquant_results

[Udaykage]

Hi after running ORFquant i got all the expected output files ex. gtf file, fasta file, final results file and tmp file but pdf/html reports not produced.

When i tried producing using the ORFquant outfiles using command

plot_ORFquant_results(for_ORFquant_file = "WHEAT.MP_for_SaTAnn",ORFquant_output_file = "WheatORFquantMP_final_ORFquant_results",annotation_file = "Novel.Triticum_aestivum.IWGSC.44_Ensemble_v1.gtf.copy.copy_Rannot",coverage_file_plus = "WHEAT.MP_coverage_plus.bedgraph", coverage_file_minus = "WHEAT.MP_coverage_minus.bedgraph")

I am getting following error: Error in cut.default(rans$delta_iso, breaks = qnt, include.lowest = T) : invalid number of intervals

I dont know whats going wrong here.

I am working on wheat and used stringtie developed gtf (reference guided annotation using gffcompare) file for mapping and preparing annotation.

[lcalviell]

Hi Uday,

Please send me a reproducible example where I can test the code on, e.g. the files you are using in this function. Is the function working if you do not specify the bedgraph files? They are used to calculate aggregate plots on splice junctions, but this step is optional.

Sorry for the inconvenience! Lorenzo

[Udaykage]

Hi Lorenzo,

Thanks for your reply. This function working fine without bedgraph files specifying, but what causing this error? Do you want me to send you the files i am using for this function?

Uday

[lcalviell]

Hi Uday, if you can that would be very helpful.

Thanks, Lorenzo

[Udaykage]

Hi Lorenzo,

I sent you all the files through cloudstor, please have look and kindly let me know whats wrong.

Thanks Uday

[lcalviell]

Dear Uday,

Thanks for the files! I looked through and found the error. You can install the newer ORFquant version (v1.02) from the github using again install_github(repo = "lcalviell/ORFquant")

and the report creation should work fine.

Best, Lorenzo

[Udaykage]

Hi Lorenzo,

Thanks for your time, its working fine now. But i dont understand these aggregate figures generated quite rightly as they are multiple figures generated, i understood red and blue color designates other and main ORF respectively, what about other colors? and why they are two figures generated for each event for example. event = up_5ss or down_3ss. I assume ss here refers to start site. To better understand can you please give some more details on this.

Also how i can separate genes with different number of ORFs and from which result file i get them?

Thanks Uday

[lcalviell]

Hi Uday, The gradient between dark blue and red indicate the presence of additional ORFs at different levels of translation. More red indicates a more complex mixture of translated ORFs mapping to those loci. Shown are alternative splice sites (ss) regions, and divided according to their 5' or 3' position (upstream or downstream with respect to the major ORF). Per each event, you will see the actual Ribo-seq coverage, and the coverage expected by the ORFquant quantification. Cases in which the coverage profiles are similar to the expected ones indicate that ORFquant was able to correctly quantify translation.

Do please check the vignette and the manual for a more comprehensive guide to explore the ORFquant output. Most of the information is present in the "_final_ORFquant_results" RData file. The "ORFs_tx" object contains transcript coordinates and quantification statistics. In addition, all the ORF coordinates, annotation and quantifications are present in the "*_Detected_ORFs.gtf" output ("CDS" field).

Do please ask me and also check our manuscript for further clarifications.

~Lorenzo

[Udaykage]

Thanks Lorenzo for your reply. May i know why is the number of protein sequences present in fasta file (~25000 sequences) are different from the number of translated ORFs detected (~48000) in my case? I assume they should be same, right?