Regarding R2Dtool metafeature plots

[PRIYANKA-22091995]

Hi,
I was trying to run the R2Dtool, the r2d liftover and r2d annotation work fine for me. However, when I try to run the r2d plotMetaTranscript script I get the following error:
Command used:
./r2d plotMetaTranscript -i "/home/priyanka/R2D_m6a_Cheui_PvSal1/annotatetrial.bed" -o "/home/priyanka/R2D_m6a_Cheui_PvSal1/metatranscript_m6A.png" --filter-field "stoichiometry" --cutoff "1" --cutoff-type "lower" -l
Error:
Arguments being passed to R2_plotMetaTranscript.R:
Rscript R2_plotMetaTranscript.R /home/priyanka/R2D_m6a_Cheui_PvSal1/annotatetrial.bed /home/priyanka/R2D_m6a_Cheui_PvSal1/metatranscript_m6A.png stoichiometry 1 lower -c loess -l
Executing R script: "/home/priyanka/R2Dtool/target/release/../../scripts/R2_plotMetaTranscript.R"
R script stdout:
[1] "Using loess method for confidence intervals."

R script stderr:
Parsed with column specification:
cols(
.default = col_double(),
transcript = col_character(),
name = col_character(),
score = col_character(),
strand = col_character(),
motif = col_character(),
gene_id = col_character(),
gene_name = col_logical(),
transcript_biotype = col_logical(),
cds_start = col_logical(),
cds_end = col_logical(),
tx_end = col_logical(),
transcript_metacoordinate = col_logical(),
abs_cds_start = col_logical(),
abs_cds_end = col_logical()
)
See spec(...) for full column specifications.
Warning message:
guess_max is a very large value, setting to 21474836 to avoid exhausting memory
Error in mutate():
ℹ In argument: interval = cut(...).
Caused by error in cut.default():
! 'x' must be numeric
Backtrace:
▆

1. ├─global compute_ratio(calls, ci_method)
2. │ └─... %>% mutate(ratio = sig / (sig + ns + 1e-09))
3. ├─dplyr::mutate(., ratio = sig/(sig + ns + 1e-09))
4. ├─tidyr::pivot_wider(...)
5. │ └─tidyr::build_wider_spec(...)
6. │ ├─tidyselect::vars_select(tbl_vars(data), !!enquo(names_from))
7. │ │ └─tidyselect:::eval_select_impl(...)
8. │ └─dplyr::tbl_vars(data)
9. │ ├─dplyr:::new_sel_vars(tbl_vars_dispatch(x), group_vars(x))
10. │ │ └─base::structure(...)
11. │ └─dplyr:::tbl_vars_dispatch(x)
12. ├─dplyr::summarise(., n = n(), .groups = "drop")
13. ├─dplyr::group_by(., interval, filter)
14. ├─dplyr::mutate(...)
15. ├─dplyr:::mutate.data.frame(...)
16. │ └─dplyr:::mutate_cols(.data, dplyr_quosures(...), by)
17. │ ├─base::withCallingHandlers(...)
18. │ └─dplyr:::mutate_col(dots[[i]], data, mask, new_columns)
19. │ └─mask$eval_all_mutate(quo)
20. │ └─dplyr (local) eval()
21. ├─base::cut(...)
22. ├─base::cut.default(...)
23. │ └─base::stop("'x' must be numeric")
24. └─base::.handleSimpleError(...)
25. └─dplyr (local) h(simpleError(msg, call))

26. └─rlang::abort(message, class = error_class, parent = parent, call = error_call)
    

Execution halted

Error: R script exited with non-zero status: ExitStatus(unix_wait_status(256))
PlotMetaTranscript generation failed. Please check the error messages above.

Please let me know what could went wrong.
Thank you in advance.

[jrkirk61]

I am getting the same exact error when I try to run this command. However, it looks like the problem actually originates with the annotate command. I can see from the code in R2_plotMetaTranscript.R that its expecting there to be numeric values for "transcript_metacoordinate", but in my annotated file I have only NAs in this column. Here is my annotate command:

r2d annotate -H -g GCF_003254395.2_Amel_HAv3.1+virus_genomic.filled.gtf -i LB_transcript_level_m6A_predictions_liftover.bed > LB_transcript_level_m6A_predictions_liftover_annotate.bed

Here is a preview of what "LB_transcript_level_m6A_predictions_liftover.bed" looks like:

chromosome      start   end     name    score   strand  transcript      start   end     name    score   strand  motif   coverage        stoichiometry   probability     fraction_modified
NC_037644.1     10937748        10937749                        -       NM_001010975.1  1005    1006    .       .       +
       TCCAATGTA       438     0.15479876160990713     0.149415        15.48
NC_037644.1     10937736        10937737                        -       NM_001010975.1  1017    1018    .       .       +
       CCAGATCAA       232     0.4913294797687861      0.84003264      49.13
NC_037644.1     10937714        10937715                        -       NM_001010975.1  1039    1040    .       .       +
       TGAAACAGT       475     0.7483660130718954      0.8618904       74.84
NC_037644.1     10937707        10937708                        -       NM_001010975.1  1046    1047    .       .       +
       GTACACAAG       447     0.32653061224489793     0.35680854      32.65
NC_037644.1     10937683        10937684                        -       NM_001010975.1  1070    1071    .       .       +
       AAATACACT       515     0.27485380116959063     0.4405879       27.49

And what "LB_transcript_level_m6A_predictions_liftover_annotate.bed" looks like:

chromosome      start   end     name    score   strand  transcript      start   end     name    score   strand  motif   coverage        stoichiometry   probability     fraction_modified       gene_id gene_name       transcript_biotype      tx_len  cds_start       cds_end tx_end  transcript_metacoordinate       abs_cds_start   abs_cds_end     up_junc_dist    down_junc_dist
NC_037644.1     10937748        10937749                        -       NM_001010975.1  1005    1006    .       .       +
       TCCAATGTA       438     0.15479876160990713     0.149415        15.48   NA      NA      NA      NA      NA      NA
      NA      NA      NA      NA      NA      NA
NC_037644.1     10937736        10937737                        -       NM_001010975.1  1017    1018    .       .       +
       CCAGATCAA       232     0.4913294797687861      0.84003264      49.13   NA      NA      NA      NA      NA      NA
      NA      NA      NA      NA      NA      NA
NC_037644.1     10937714        10937715                        -       NM_001010975.1  1039    1040    .       .       +
       TGAAACAGT       475     0.7483660130718954      0.8618904       74.84   NA      NA      NA      NA      NA      NA
      NA      NA      NA      NA      NA      NA
NC_037644.1     10937707        10937708                        -       NM_001010975.1  1046    1047    .       .       +
       GTACACAAG       447     0.32653061224489793     0.35680854      32.65   NA      NA      NA      NA      NA      NA
      NA      NA      NA      NA      NA      NA
NC_037644.1     10937683        10937684                        -       NM_001010975.1  1070    1071    .       .       +
       AAATACACT       515     0.27485380116959063     0.4405879       27.49   NA      NA      NA      NA      NA      NA
      NA      NA      NA      NA      NA      NA

Any help someone can offer would be appreciated! Thank you!