hi
I am using riboSeqR to analyze ribosome profling data. However, after framecounting, all the frame counts are 0.
Here is what I have done from the beginning. I removed the linker sequence and timmed the reads first, then align all the reads to ncRNA.fq and collect unaligned reads. Then I used bowtie to align all the norrna reads to human transcriptome. After alignment, I used riboSeqR.
Below is my R console. Anyone can tell me what is going on? I will really appreciate.
> library("riboSeqR")
> humanFasta <- paste("/Users/yingying/Desktop/Johnlab/Exps/Exp1/humancdna.fa")
> fastaCDS <- findCDS(fastaFile=humanFasta,startCodon=c("ATG"),stopCodon=c("TAG","TAA","TGA"))
Read 5174699 items
> ribofiles <- paste("/Users/yingying/Desktop/Johnlab/Exps/Exp1/A549Con.bowtie.out")
> riboDat <- readRibodata ( ribofiles, replicates=c("con"))
Reading ribosomal files....done!
> fCs <- frameCounting(riboDat, fastaCDS, lengths=24:33)
Calling frames..........done!
> 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
> fastaCDS
GRanges object with 2865412 ranges and 6 metadata columns:
seqnames ranges strand | frame startCodon stopCodon context minus3 plus1
<Rle> <IRanges> <Rle> | <numeric> <character> <character> <character> <character> <character>
[1] ENST00000603326.1 [ 6, 19] * | 2 ATG TAC ACTATGG A G
[2] ENST00000390583.1 [ 9, 31] * | 2 ATG AAC ACTATGG A G
[3] ENST00000390575.1 [13, 20] * | 0 ATG TAC GCTATGG G G
[4] ENST00000390571.1 [ 9, 31] * | 2 ATG TAC ACTATGA A A
[5] ENST00000390588.1 [13, 20] * | 0 ATG TAC GCTATGG G G
... ... ... ... . ... ... ... ... ... ...
[2865408] ENST00000638565.1 [ 872, 892] * | 1 ATG TGA CAGATGC C C
[2865409] ENST00000638565.1 [1102, 1113] * | 0 ATG TAA AAAATGC A C
[2865410] ENST00000638565.1 [1162, 1185] * | 0 ATG TGA CTGATGA C A
[2865411] ENST00000638565.1 [1294, 1331] * | 0 ATG TGA CAAATGC C C
[2865412] ENST00000638565.1 [1328, 1331] * | 1 ATG TGA CCCATGA C A
-------
seqinfo: 180869 sequences from an unspecified genome; no seqlengths
> readRibodata ( ribofiles, replicates=c("con"))
Reading ribosomal files....done!
An object of class "riboData"
Slot "riboGR":
$A549Con.bowtie.out
GRanges object with 621370 ranges and 0 metadata columns:
seqnames ranges strand
<Rle> <IRanges> <Rle>
[1] ENST00000265620.11 [152, 181] +
[2] ENST00000592420.1 [129, 157] +
[3] ENST00000620941.1 [447, 475] +
[4] ENST00000616743.1 [395, 423] +
[5] ENST00000573216.5 [ 68, 96] +
... ... ... ...
[621366] ENST00000392730.2 [433, 462] +
[621367] ENST00000571730.1 [ 19, 48] +
[621368] ENST00000351989.7 [ 43, 71] +
[621369] ENST00000368984.7 [170, 198] +
[621370] ENST00000377698.3 [750, 777] +
-------
seqinfo: 63130 sequences from an unspecified genome; no seqlengths
Slot "rnaGR":
list()
Slot "replicates":
[1] con
Levels: con
Thanks a lot!
Yingying