Question: error: NA counts not permitted
gravatar for clarisbaby
19 months ago by
clarisbaby0 wrote:
hey everyone,
I am trying to run the following code for C. elegans paired end data using the following code:

library("biomartr", lib.loc="~/R/x86_64-pc-linux-gnu-library/3.4")
export(Celegans, "dataset/ce11.fasta", compress=FALSE)
getGFF(db = "ensembl", organism = "Caenorhabditis elegans", reference = TRUE, path = file.path("dataset"))
system("gunzip dataset/Caenorhabditis_elegans.WBcel235.91_ensembl.gff3.gz")
args <- systemArgs(sysma="./param/tophat.param", mytargets="targetsPE.txt") 
system("bowtie2-build ./dataset/ce11.fasta ./dataset/ce11.fasta")
bampaths <- runCommandline(args=args)
read_statsDF <- alignStats(args)
write.table(read_statsDF, "results/alignStats.xls", row.names=FALSE, quote=FALSE, sep="\t")
txdb <- makeTxDbFromGFF(file="dataset/Caenorhabditis_elegans.WBcel235.91_ensembl.gff3.gz", format="gff")
saveDb(txdb, file="dataset/database.sqlite")
txdb <- loadDb("./dataset/database.sqlite")
eByg <- exonsBy(txdb, by=c("tx"))
countDFeByg <- assays(counteByg)$counts
countDFeByg <-
countDFeByg[] <- 0
write.table(countDFeByg, "results/countDFeByg.xls", col.names=NA, quote=FALSE, sep="\t")
countDF <- read.table("results/countDFeByg.xls")
cmp <- readComp(args, format="matrix", delim="-")
edgeDF <- run_edgeR(countDF=countDF, targets=targetsin(args), cmp=cmp[[1]], independent=FALSE, mdsplot="")
write.table(edgeDF, "results/edgeRcomp.xls", quote=FALSE, sep="\t", col.names = NA)
edgeDF <- read.table("./results/edgeRcomp.xls")
DEG_list <- filterDEGs(degDF=edgeDF, filter, plot = FALSE)
while running the following code i am facing the error as below
     Error in calcNormFactors.default(object = object$counts, lib.size = object$samples$lib.size,  : 
     NA counts not permitted
     Called from: calcNormFactors.default(object = object$counts, lib.size = object$samples$lib.size,
     method = method, refColumn = refColumn, logratioTrim = logratioTrim, sumTrim = sumTrim, 
     doWeighting = doWeighting, Acutoff = Acutoff, p = p)

I have already tried doing 

countDFeByg[] <- 0

which is said to remove the NA.

It would be very kind if someone may help me to solve the error or point out where I am going wrong

Thanks in advance.



edger systempiper rna-seq • 307 views
ADD COMMENTlink modified 19 months ago by Aaron Lun25k • written 19 months ago by clarisbaby0
Answer: error: NA counts not permitted
gravatar for Aaron Lun
19 months ago by
Aaron Lun25k
Cambridge, United Kingdom
Aaron Lun25k wrote:

Obviously, whatever matrix is being passed to calcNormFactors has NA counts. Removing NAs in countDFeByg won't help if the NAs are being introduced afterwards.

  • Are the NAs present in countDF? What does say?
  • Do any of the libraries have zero counts? This will result in NAs if run_edgeR() computes CPMs.

Otherwise, it's something happening inside run_edgeR() (I assume this is where the error is being raised), and you'll have to do debug(run_edgeR) to see how the NAs are being introduced.

ADD COMMENTlink modified 19 months ago • written 19 months ago by Aaron Lun25k

anyNA(countDF) says false...

countDFeByg has all the values as zero. When i started looking around i found that it may be due to BAM and GFF files that use different chromosome names.

how can i solve that..

my gff file looks as below

##gff-version 3
##sequence-region   I 1 15072434
##sequence-region   II 1 15279421
##sequence-region   III 1 13783801
##sequence-region   IV 1 17493829
##sequence-region   MtDNA 1 13794
##sequence-region   V 1 20924180
##sequence-region   X 1 17718942
#!genome-build WormBase WBcel235

where as the alignment file looks as

@HD VN:1.0 SO:coordinate

@SQ SN:chrI LN:15072434

@SQ SN:chrII LN:15279421

@SQ SN:chrIII LN:13783801

@SQ SN:chrIV LN:17493829

@SQ SN:chrM LN:13794

@SQ SN:chrV LN:20924180

@SQ SN:chrX LN:17718942

@PG ID:TopHat VN:2.1.0


Thanking you in advance.

ADD REPLYlink written 19 months ago by clarisbaby0

Well, obviously, that's going to be a problem. The solution is simple. Fix your GFF file so that the chromosome names have chr in front of them (or in the case of MtDNA, replaced with chrM). rtracklayer has a few commands for importing GFF files; you should be able to edit the sequence names, and then export it back to another file.

ADD REPLYlink written 19 months ago by Aaron Lun25k
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