Hi,

I have analysed ribo-seq and rna-seq data from wild type or mutant mice tissue using riboSeqR.

But analysis failed at calling or reading frame step and all frame counts fell into 0.

My R console looks like this (only analysed WT data):

> library(riboSeqR)
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, do.call, 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, unsplit

Loading required package: S4Vectors
Loading required package: stats4
Creating a generic function for ‘nchar’ from package ‘base’ in package ‘S4Vectors’
Loading required package: IRanges
Loading required package: GenomeInfoDb
Loading required package: abind

> datadir <- system.file("extdata", package = "riboSeqR")
> musFasta <- paste(datadir, "/refMrna.fa", sep = "")
> fastaCDS <- findCDS(fastaFile = musFasta, startCodon = c("ATG"), stopCodon = c("TAG", "TAA", "TGA"))
Read 2033198 items

> rnafiles <- paste(datadir, "/rna1", sep = "")
> ribofiles <- paste(datadir, "/ribo1", sep = "")
> riboDat <- readRibodata(ribofiles, replicates = c("WT"))
Reading ribosomal files....done!
> fCs <- frameCounting(riboDat, fastaCDS)
Calling frames..........done!
There were 15 warnings (use warnings() to see them)
> warnings()
Warning messages:
1: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
2: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
3: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
4: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
5: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
6: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
7: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
8: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
9: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
10: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
11: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
12: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
13: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
14: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
15: In `levels<-`(`*tmp*`, value = if (nl == nL) as.character(labels) else paste0(labels,  ... :
  duplicated levels in factors are deprecated
> fS <- readingFrame(rC = fCs); fS
         26 27 28 29 30
          0  0  0  0  0
          0  0  0  0  0
          0  0  0  0  0
frame.ML  0  0  0  0  0

> sessionInfo()
R version 3.2.2 (2015-08-14)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.11.1 (El Capitan)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] riboSeqR_1.2.0       abind_1.4-3          GenomicRanges_1.20.8 GenomeInfoDb_1.4.3   IRanges_2.2.9       
[6] S4Vectors_0.6.6      BiocGenerics_0.14.0 

loaded via a namespace (and not attached):
[1] XVector_0.8.0

The refMrna.fa is mouse transcriptome reference from following website ( http://hgdownload.soe.ucsc.edu/goldenPath/mm9/bigZips/refMrna.fa.gz ). And the ribo1 and rna1 files are bowtie outputs mapped against mouse transcriptome using --supress 1,6,7,8 option.

the analysis using test chlamy files in a folder extdata is succesful.

Could anyone tell me some advice or information about this error ?

Thanks a lot.

Tak 

Thanks Sonali,

I checked my fastaCDS and riboDat objects as shown below.

> riboDat
An object of class "riboData"
Slot "riboGR":
$E2ribo1
GRanges object with 76480065 ranges and 0 metadata columns:
              seqnames       ranges strand
                 <Rle>    <IRanges>  <Rle>
         [1] NM_028136 [1803, 1831]      +
         [2] NR_004412 [ 117,  142]      +
         [3] NR_024200 [ 117,  142]      +
         [4] NR_004411 [ 116,  141]      +
         [5] NR_004413 [ 117,  142]      +
         ...       ...          ...    ...
  [76480061] NR_004411 [ 117,  141]      +
  [76480062] NR_004413 [ 118,  142]      +
  [76480063] NM_009530 [4248, 4275]      +
  [76480064] NM_009272 [ 245,  271]      +
  [76480065] NM_207523 [ 379,  406]      +
  -------
  seqinfo: 28006 sequences from an unspecified genome; no seqlengths

Slot "rnaGR":
list()

Slot "replicates":
[1] WT
Levels: WT

> fastaCDS
GRanges object with 967022 ranges and 1 metadata column:
                 seqnames      ranges strand   |     frame
                    <Rle>   <IRanges>  <Rle>   | <numeric>
       [1] NM_001289633 1 [ 54, 2762]      *   |         2
       [2] NM_001289633 1 [145,  267]      *   |         0
       [3] NM_001289633 1 [313,  321]      *   |         0
       [4] NM_001289633 1 [523,  543]      *   |         0
       [5] NM_001289633 1 [652,  753]      *   |         0
       ...            ...         ...    ... ...       ...
  [967018]    NM_023456 3  [386, 451]      *   |         1
  [967019]    NM_023456 3  [393, 548]      *   |         2
  [967020]    NM_023456 3  [478, 580]      *   |         0
  [967021]    NM_023456 3  [533, 544]      *   |         1
  [967022]    NM_023456 3  [555, 580]      *   |         2
  -------
  seqinfo: 34706 sequences from an unspecified genome; no seqlengths

As your information, sequence names are slightly different in these two objects (Refseq accession or Refseq accession including version number).

Now I'm trying to remove these hyphens and version numbers in fastaCDS. 

Thanks a lot for your information, really helpful!

Tak