Error in frameCounting using riboSeqR
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Entering edit mode
@yingyingguo0228-13829
Last seen 7.2 years ago

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

riboseq R riboseqr • 1.5k views
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Entering edit mode
@thomas-j-hardcastle-3860
Last seen 7.1 years ago
United Kingdom

Hi Yingying,

This almost always happens because of a mismatch between the headers in the fasta file and the gene names in the bowtie file - which in turn, almost always happens because bowtie drops everything after the first space in the header file, whereas riboSeqR uses the full header. I suggest you take a look at the fasta file and see if you can update it so that it uses the same names as the bowtie output.

Best wishes,

Tom

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Entering edit mode
@yingyingguo0228-13829
Last seen 7.2 years ago

Hi Tom,

Thank you for your reply!

At the begining, I found people having the same problems as me. They fixed their problem by making header name of their fastq file and bowtie output the same. so I checked mine and noticed that the header of my fastaq file is different from bowtie output.  So I used command

"sed 's/cdna.*$//' Homo_sapiens.GRCh38.cdna.all.fa > humancdna.fa"  to remove all the information after the first space in header of fastq. 

Here is the old header of fastq file. 

>ENST00000448914.1 cdna chromosome:GRCh38:14:22449113:22449125:1 gene:ENSG00000228985.1 gene_biotype:TR_D_gene transcript_biotype:TR_D_gene gene_symbol:TRDD3 description:T-cell receptor delta diversity 3 [Source:HGNC Symbol;Acc:HGNC:12256]​

after sed, the header is 

>ENST00000448914.1​

This is the same as my bowtie output.

However, it still gives me 0 for framecounting. 

Did I do something wrong?  

I really appreciate for your help 

Best,

Yingying

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Entering edit mode
@yingyingguo0228-13829
Last seen 7.2 years ago

Hi Tom,

I just tried again. It turns out that there is a blank space after each header of fastq file. So I should use command

"sed 's/ cdna.*$//' Homo_sapiens.GRCh38.cdna.all.fa > humancdna.fa"​ instead of 

"sed 's/cdna.*$//' Homo_sapiens.GRCh38.cdna.all.fa > humancdna.fa"​. 

I did not realize that this blank space would cause such a big difference. 

Thank you so much for your suggestion! I am new to bioinformatic and this is the first time I post question here. I did not expect that you reply so fast. Thanks agian!

Best,

Yingying

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