Error in apply(counts, 2, function(x) rpkm(x, lengths)) : dim(X) must have a positive length

Question

[shail.nair05]

I am trying to convert featurecounts output (raw reads of transcripts) to TPM via tpm_rpkm.R(https://github.com/andysaurin/tpm_rpkm) script. but i am getting error saying Error in apply(counts, 2, function(x) rpkm(x, lengths)) : dim(X) must have a positive length

Here is the script

#! /usr/bin/env Rscript

# Author: Andy Saurin (--------------------)
#
# Simple RScript to calculate RPKMs and TPMs
# based on method for RPKM/TPM calculations shown in http://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/
#
# The input file is the output of featureCounts
#

rpkm <- function(counts, lengths) {
  pm <- sum(counts) /1e6
  rpm <- counts/pm
  rpm/(lengths/1000)
}

tpm <- function(counts, lengths) {
  rpk <- counts/(lengths/1000)
  coef <- sum(rpk) / 1e6
  rpk/coef
}


## read table from featureCounts output
args <- commandArgs(T)

tag <- tools::file_path_sans_ext(args[1])


cat('Reading in featureCounts data...')
ftr.cnt <- read.table(args[1], sep="\t", header=T, quote="") #Important to disable default quote behaviour or else genes with apostrophes will be taken as strings
cat(' Done\n')

if ( ncol(ftr.cnt) < 7 ) { 
    cat(' The input file is not the raw output of featureCounts (number of columns > 6) \n')
    quit('no')
}

lengths = ftr.cnt[,6]

counts <- ftr.cnt[,7:ncol(ftr.cnt)]

cat('Performing RPKM calculations...')

rpkms <- apply(counts, 2, function(x) rpkm(x, lengths) )
ftr.rpkm <- cbind(ftr.cnt[,1:6], rpkms)

rpkms <- apply(counts, 2, function(x) rpkm(x, lengths) )
ftr.rpkm <- cbind(ftr.cnt[,1:6], rpkms)
write.table(ftr.rpkm, file=paste0(tag, "_rpkm.txt"), sep="\t", row.names=FALSE, quote=FALSE)
cat(' Done.\n\tSaved as ')
cat ( paste0(tag, "_rpkm.txt", '\n') )

cat('Performing TPM calculations...')

tpms <- apply(counts, 2, function(x) tpm(x, lengths) )

ftr.tpm <- cbind(ftr.cnt[,1:6], tpms)

write.table(ftr.tpm, file=paste0(tag, "_tpm.txt"), sep="\t", row.names=FALSE, quote=FALSE)
cat(' Done.\n\tSaved as ')
cat ( paste0(tag, "_tpm.txt", '\n') )


quit('no')




**command output**

Rscript tpm_rpkm.R 450-3-hard_filtered.featureCounts
Reading in featureCounts data... Done
Performing RPKM calculations...Error in apply(counts, 2, function(x) rpkm(x, lengths)) :
dim(X) must have a positive length
halt execution

My featurecount table looks like this

Geneid | Chr | Start | End | Strand | Length |
1_1 | NODE_1_length_59711_cov_84.026979_g0_i0 | 116 | 904 | + | 789 | 198
1_2 | NODE_1_length_59711_cov_84.026979_g0_i0 | 1178 | 3514 | - | 2337 | 2294
1_3 | NODE_1_length_59711_cov_84.026979_g0_i0 | 3618 | 4319 | + | 702 | 502
1_4 | NODE_1_length_59711_cov_84.026979_g0_i0 | 4337 | 4921 | + | 585 | 320
1_5 | NODE_1_length_59711_cov_84.026979_g0_i0 | 4953 | 5906 | + | 954 | 799
1_6 | NODE_1_length_59711_cov_84.026979_g0_i0 | 5920 | 7056 | + | 1137 | 532
1_7 | NODE_1_length_59711_cov_84.026979_g0_i0 | 7061 | 8071 | + | 1011 | 761
1_8 | NODE_1_length_59711_cov_84.026979_g0_i0 | 8068 | 8766 | + | 699 | 188
1_9 | NODE_1_length_59711_cov_84.026979_g0_i0 | 8766 | 9656 | + | 891 | 217
1_10 | NODE_1_length_59711_cov_84.026979_g0_i0 | 9640 | 10710 | + | 1071 | 408
1_11 | NODE_1_length_59711_cov_84.026979_g0_i0 | 10692 | 11348 | + | 657 | 162
1_12 | NODE_1_length_59711_cov_84.026979_g0_i0 | 11359 | 12282 | + | 924 | 342

Does anyone know how to deal with this?

[rpolicastro]

tr.cnt <- read.table(args[1], sep="\t", header=T, quote="")

should be

tr.cnt <- read.table(args[1], sep="|", header=T, quote="", strip.white=TRUE)

[shail.nair05]

That did not work. It threw error saying

The input file is not the raw output of featureCounts (number of columns > 6)

But i got the solution from https://stackoverflow.com/questions/66762378/error-in-applycounts-2-functionx-rpkmx-lengths-dimx-must-have-a-pos

Updating the counts <- ftr.cnt[,7:ncol(ftr.cnt), drop=FALSE]

worked smoothly.

Thanks

[Ram]

Posting the same question on multiple forums is bad etiquette. Even if you do cross-posts, it is common decency to mention your cross-posts so people don't invest effort when you've already been helped elsewhere.

[shail.nair05]

Sorry for that. will keep in mind next time. thanks