DEXSeq Error loading in data set - subscript out of bound

Question

[osiemen]

Hi I am currently following the tutorial of DEXSeq to do a differential Exon usage analysis on my dataset. I am currently at the step where I read my dataset in to R to construct a DEXSeqDataSet using the function DEXSeqDataSetFromHTSeq() .

dxd = DEXSeqDataSetFromHTSeq(
   countFiles,
   sampleData=sampleTable,
   design= ~ sample + exon + condition:exon,
   flattenedfile=flattenedFile )

I have total 4 countFiles which I constructed from my actual dataset to apply this function on a smaller dataset. The countFiles look like following:

RNA1_test.txt:

""ENSG00000000003.14"":"001"    36
""ENSG00000000003.14"":"002"    183
""ENSG00000000003.14"":"003"    45
""ENSG00000000003.14"":"004"    0
""ENSG00000000003.14"":"005"    32
""ENSG00000000003.14"":"006"    21
""ENSG00000000003.14"":"007"    22
""ENSG00000000003.14"":"008"    24
""ENSG00000000003.14"":"009"    26
""ENSG00000000003.14"":"010"    17
""ENSG00000000003.14"":"011"    34
""ENSG00000000003.14"":"012"    10
""ENSG00000000003.14"":"013"    12
""ENSG00000000003.14"":"014"    7
""ENSG00000000003.14"":"015"    2
""ENSG00000000003.14"":"016"    0
""ENSG00000000003.14"":"017"    0

RNA2_test.txt:

""ENSG00000000003.14"":"001"    34
""ENSG00000000003.14"":"002"    191
""ENSG00000000003.14"":"003"    60
""ENSG00000000003.14"":"004"    0
""ENSG00000000003.14"":"005"    41
""ENSG00000000003.14"":"006"    36
""ENSG00000000003.14"":"007"    42
""ENSG00000000003.14"":"008"    50
""ENSG00000000003.14"":"009"    47
""ENSG00000000003.14"":"010"    26
""ENSG00000000003.14"":"011"    38
""ENSG00000000003.14"":"012"    5
""ENSG00000000003.14"":"013"    18
""ENSG00000000003.14"":"014"    7
""ENSG00000000003.14"":"015"    1
""ENSG00000000003.14"":"016"    1
""ENSG00000000003.14"":"017"    0

etc...

The amount of lines the transcripts/Exons in the the 4 countFiles are identical only the counts are different. I also used a sample of the actual flattened gff file that was used for the counting the reads :

chrX    dexseq_prepare_annotation.py    aggregate_gene  100627109       100639991       .       -       .       gene_id ""ENSG00000000003.14""
chrX    dexseq_prepare_annotation.py    exonic_part     100627109       100628669       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000612152.4""; exonic_part_number "001"
chrX    dexseq_prepare_annotation.py    exonic_part     100628670       100629986       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000612152.4""; exonic_part_number "002"
chrX    dexseq_prepare_annotation.py    exonic_part     100630759       100630866       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000612152.4""; exonic_part_number "003"
chrX    dexseq_prepare_annotation.py    exonic_part     100632063       100632068       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4""; exonic_part_number "004"
chrX    dexseq_prepare_annotation.py    exonic_part     100632485       100632540       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000614008.4""; exonic_part_number "005"
chrX    dexseq_prepare_annotation.py    exonic_part     100632541       100632568       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000496771.5"+"ENST00000614008.4""; exonic_part_number "006"
chrX    dexseq_prepare_annotation.py    exonic_part     100633405       100633441       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000496771.5"+"ENST00000614008.4"+"ENST00000612152.4""; exonic_part_number "007"
chrX    dexseq_prepare_annotation.py    exonic_part     100633442       100633539       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4"+"ENST00000612152.4"+"ENST00000494424.1"+"ENST00000496771.5"+"ENST00000373020.8""; exonic_part_number "008"
chrX    dexseq_prepare_annotation.py    exonic_part     100633931       100634029       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4"+"ENST00000612152.4"+"ENST00000494424.1"+"ENST00000496771.5"+"ENST00000373020.8""; exonic_part_number "009"
chrX    dexseq_prepare_annotation.py    exonic_part     100635178       100635252       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4"+"ENST00000612152.4"+"ENST00000494424.1"+"ENST00000496771.5"+"ENST00000373020.8""; exonic_part_number "010"
chrX    dexseq_prepare_annotation.py    exonic_part     100635558       100635746       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4"+"ENST00000612152.4"+"ENST00000494424.1"+"ENST00000496771.5"+"ENST00000373020.8""; exonic_part_number "011"
chrX    dexseq_prepare_annotation.py    exonic_part     100636191       100636607       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000496771.5""; exonic_part_number "012"
chrX    dexseq_prepare_annotation.py    exonic_part     100636608       100636689       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000496771.5""; exonic_part_number "013"
chrX    dexseq_prepare_annotation.py    exonic_part     100636690       100636792       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8""; exonic_part_number "014"
chrX    dexseq_prepare_annotation.py    exonic_part     100636793       100636806       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000373020.8"+"ENST00000614008.4"+"ENST00000494424.1"+"ENST00000612152.4""; exonic_part_number "015"
chrX    dexseq_prepare_annotation.py    exonic_part     100636807       100637104       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000614008.4"+"ENST00000494424.1"+"ENST00000612152.4""; exonic_part_number "016"
chrX    dexseq_prepare_annotation.py    exonic_part     100639945       100639991       .       -       .       gene_id ""ENSG00000000003.14""; transcripts ""ENST00000494424.1""; exonic_part_number "017"

However when I run my code :

#get count file names
countFiles= list.files(path = "Desktop",pattern = "RNA", full.names = TRUE)
basename(countFiles)

[1] "RNA1_test.txt" "RNA2_test.txt" "RNA3_test.txt" "RNA4_test.txt"
#get gff file name
 flattenedFile = list.files(path = "Desktop", pattern="gff$", full.names=TRUE)
basename(flattenedFile)
[1] "dexseq_test.gff"

sampleTable = data.frame(
  row.names = c( "retina1", "retina2", 
                 "brain1", "brain2"  ),
  condition = c("experiment", "experiment",
                "control", "control" ))

dxd = DEXSeqDataSetFromHTSeq(
countFiles,
sampleData=sampleTable2,
design= ~ sample + exon + condition:exon,
flattenedfile=flattenedFile )

I receive the following error:

Error in FUN(X[[i]], ...) : subscript out of bound

I am not sure what might cause this error, since the gene ids in the gff file also match the ones used in my countFiles. Anyone familiar with this error?

[osiemen]

One of the differences I noticed between my files and the files used in the example are the quotes " " in the countFiles -> ""ENSG00000000003.14"":"001" 34 , but im not sure if that could cause it

Answer 1

[uwe.schwartz]

Had the same problem. Removing the quotes from the count table did the job.

[osiemen]

Indeed thats what I had to do as well!

[coboyfan12]

Worked for me too! Ty!