Error at Creating Count Table for DESeq2

[sazz]

I have used Tophat-CuffDiff pipeline so far but I want to give a try for DESeq2. I have 2 conditions and 3 replicates for each, aim is to find the differentially expressed genes.

For a couple of days, I am trying to use HTSeq to prepare my count files. I guess I did it but now I am stuck at creating the count table as the DESeq2 input.

I didn't use R that much so far, so I am having difficulties. Here is the problem:

> library('DESeq2')
Loading required package: GenomicRanges
Loading required package: BiocGenerics
Loading required package: parallel

Attaching package: ‘BiocGenerics’

The following objects are masked from ‘package:parallel’:

    clusterApply, clusterApplyLB, clusterCall, clusterEvalQ, clusterExport, clusterMap, parApply, parCapply,
    parLapply, parLapplyLB, parRapply, parSapply, parSapplyLB

The following object is masked from ‘package:stats’:

    xtabs

The following objects are masked from ‘package:base’:

    anyDuplicated, append, as.data.frame, as.vector, cbind, colnames, duplicated, eval, evalq, Filter, Find,
    get, intersect, is.unsorted, lapply, Map, mapply, match, mget, order, paste, pmax, pmax.int, pmin,
    pmin.int, Position, rank, rbind, Reduce, rep.int, rownames, sapply, setdiff, sort, table, tapply, union,
    unique, unlist

Loading required package: IRanges
Loading required package: XVector
Loading required package: Rcpp
Loading required package: RcppArmadillo

> setwd("C:/Python27/SKMEL-5")
> directory<-"C:/Python27/SKMEL-5/ALL"
> sampleFiles <- grep("SKMEL-5",list.files(directory),value=TRUE)
> sampleCondition<-c("KD","KD","KD","WT","WT","WT")
> sampleTable<-data.frame(sampleName=sampleFiles, fileName=sampleFiles, condition=sampleCondition)
> sampleTable
       sampleName        fileName condition
1 SKMEL-5_I-1.txt SKMEL-5_I-1.txt        KD
2 SKMEL-5_I-2.txt SKMEL-5_I-2.txt        KD
3 SKMEL-5_I-3.txt SKMEL-5_I-3.txt        KD
4 SKMEL-5_L-1.txt SKMEL-5_L-1.txt        WT
5 SKMEL-5_L-2.txt SKMEL-5_L-2.txt        WT
6 SKMEL-5_L-3.txt SKMEL-5_L-3.txt        WT
> ddsHTSeq<-DESeqDataSetFromHTSeqCount(sampleTable=sampleTable, directory=directory, design=~condition)
Error in DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory,  : 
  Gene IDs (first column) differ between files.
In addition: There were 36 warnings (use warnings() to see them)

Here is the 36 warnings:

Warning messages:
1: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
2: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
3: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
4: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
5: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
6: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input
7: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
8: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
9: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
10: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
11: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
12: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input
13: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
14: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
15: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
16: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
17: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
18: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input
19: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
20: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
21: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
22: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
23: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
24: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input
25: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
26: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
27: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
28: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
29: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
30: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input
31: In read.table(file.path(directory, fn)) :
  line 1 appears to contain embedded nulls
32: In read.table(file.path(directory, fn)) :
  line 2 appears to contain embedded nulls
33: In read.table(file.path(directory, fn)) :
  line 3 appears to contain embedded nulls
34: In read.table(file.path(directory, fn)) :
  line 4 appears to contain embedded nulls
35: In read.table(file.path(directory, fn)) :
  line 5 appears to contain embedded nulls
36: In scan(file = file, what = what, sep = sep, quote = quote,  ... :
  embedded nul(s) found in input

Because it says "Gene IDs (first column) differ between files.", I have checked each file but all have the same number of rows and I guess the first column is same for all (well, I have used the same gtf file for all of them, so it must be).

I know the problem is at a very basic stage but I have no clue as an R-noob.

[sazz]

Solved, my files were not in Tab Delimited format :/

[angus878]

I got the same issue and found your post helpful. To solve I opened the file in notepad and changed the encoding from Unicode to ANSI and then it imported cleanly into R.