Question

RIPSeeker ripSeek does not produce any output

0

Entering edit mode

ofonov ▴ 10

@ofonov-12623

Last seen 7.1 years ago

Norway/Oslo

I am having troubles getting results with RIPSeeker vignette example. I have installed all dependency packages, and use code from the vignette. plotCoverage works as expected, however main functionality - ripSeek does not output any result.

library(RIPSeeker)
extdata.dir <- system.file("extdata", package="RIPSeeker")
bamFiles <- list.files(extdata.dir, "\\.bam$", recursive=TRUE, full.names=TRUE)
bamFiles <- grep("PRC2", bamFiles, value=TRUE)
bamFiles
ripGal <- combineAlignGals(grep(pattern="SRR039214", bamFiles, value=TRUE, invert=TRUE),reverseComplement=TRUE, genomeBuild="mm9")
ctlGal <- combineAlignGals(grep(pattern="SRR039214", bamFiles, value=TRUE, invert=FALSE),reverseComplement=TRUE, genomeBuild="mm9")
ripGal
ctlGal

###################################################
### code chunk number 5: plotCoverage
###################################################
ripGR <- as(ripGal, "GRanges")
ripGRList <- split(ripGR, seqnames(ripGR))
# get only the nonempty chromosome
ripGRList <- ripGRList[elementNROWS(ripGRList) != 0]
ctlGR <- as(ctlGal, "GRanges")
ctlGRList <- GRangesList(as.list(split(ctlGR, seqnames(ctlGR))))
ctlGRList <- ctlGRList[lapply(ctlGRList, length) != 0]
binSize <- 1000
par(mfrow=c(1, 2), mar=c(2, 2, 2, 0) + 0.1)
tmp <- lapply(ripGRList, plotStrandedCoverage, binSize=binSize,
              xlab="", ylab="", plotLegend=TRUE,
              legend.cex=1.5, main="RIP", cex.main=1.5)
tmp <- lapply(ctlGRList, plotStrandedCoverage, binSize=binSize,
              xlab="", ylab="", plotLegend=TRUE,
              legend.cex=1.5, main="CTL", cex.main=1.5)

###############################################################
# ripSeek: the all-in-one function
###############################################################
# specify control name
cNAME <- "SRR039214"

# output file directory
outDir <- file.path(getwd(), "RIPSeeker_vigenette_example_PRC2")

# Parameters setting
binSize <- NULL      # set to NULL to automatically determine bin size
minBinSize <- 10000  # min bin size in automatic bin size selection
maxBinSize <- 10100     # max bin size in automatic bin size selection
multicore <- TRUE         # use multicore
strandType <- "-"         # set strand type to minus strand

biomart <- "ENSEMBL_MART_ENSEMBL"           # use archive to get ensembl 65
biomaRt_dataset <- "mmusculus_gene_ensembl" # mouse dataset id name
host <- "dec2011.archive.ensembl.org"       # use ensembl 65 for annotation for mm9
goAnno <- "org.Mm.eg.db"                    # GO annotation database

################ run main function ripSeek to predict RIP ################
seekOut.PRC2 <- ripSeek(bamPath = bamFiles, cNAME = cNAME,
                   reverseComplement = TRUE, genomeBuild = "mm9",
                   strandType = strandType,
                   uniqueHit = TRUE, assignMultihits = TRUE,
                   rerunWithDisambiguatedMultihits = TRUE,
                   binSize=binSize, minBinSize = minBinSize,
                   maxBinSize = maxBinSize,
                   biomart=biomart, host=host,
                   biomaRt_dataset = biomaRt_dataset, goAnno = goAnno,
                   multicore=multicore, outDir=outDir)

names(seekOut.PRC2)

I get following output running ripSeek function:

*I. Collect alignment files

RIP alignment files:
    /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039210_processed_tophat/accepted_hits_noDup_sel_chrX.bam
    /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039211_processed_tophat/accepted_hits_noDup_sel_chrX.bam

Control alignment files:
    /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039214_processed_tophat/accepted_hits_noDup_sel_chrX.bam


*II. Analyzing RIP library:

**A. Process and combine alignment files

Processing /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039210_processed_tophat/accepted_hits_noDup_sel_chrX.bam ... All hits are returned with flags.
Processing /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039211_processed_tophat/accepted_hits_noDup_sel_chrX.bam ... All hits are returned with flags.
2 BAM files are combined
*** Only reads from strand - will be considered.
*** Only unique hits are used to compute read count.
*** chr1 do not have any alignment.
*** chr10 do not have any alignment.
*** chr11 do not have any alignment.
*** chr12 do not have any alignment.
*** chr13 do not have any alignment.
*** chr14 do not have any alignment.
*** chr15 do not have any alignment.
*** chr16 do not have any alignment.
*** chr17 do not have any alignment.
*** chr18 do not have any alignment.
*** chr19 do not have any alignment.
*** chr2 do not have any alignment.
*** chr3 do not have any alignment.
*** chr4 do not have any alignment.
*** chr5 do not have any alignment.
*** chr6 do not have any alignment.
*** chr7 do not have any alignment.
*** chr8 do not have any alignment.
*** chr9 do not have any alignment.
*** chrM do not have any alignment.
*** chrY do not have any alignment.

*** 21 pseudoreads are appended to the end.

**B. Run NB HMM on each chromosome

**C. Disambiguate multihits based on posterior

2075/12375 multihit reads corresponding to 4871 ambiguous alignments
have been assigned to 2075 unique regions with maximum posterior for the enriched state

**D. Re-run NB HMM with unique hits + disambiguated multihits.

*** chr1 do not have any alignment.
*** chr10 do not have any alignment.
*** chr11 do not have any alignment.
*** chr12 do not have any alignment.
*** chr13 do not have any alignment.
*** chr14 do not have any alignment.
*** chr15 do not have any alignment.
*** chr16 do not have any alignment.
*** chr17 do not have any alignment.
*** chr18 do not have any alignment.
*** chr19 do not have any alignment.
*** chr2 do not have any alignment.
*** chr3 do not have any alignment.
*** chr4 do not have any alignment.
*** chr5 do not have any alignment.
*** chr6 do not have any alignment.
*** chr7 do not have any alignment.
*** chr8 do not have any alignment.
*** chr9 do not have any alignment.
*** chrM do not have any alignment.
*** chrY do not have any alignment.

*** 21 pseudoreads are appended to the end.

*II.2. Analyzing control library:

**A. Process and combine alignment files

Processing /Library/Frameworks/R.framework/Versions/3.3/Resources/library/RIPSeeker/extdata/PRC2/SRR039214_processed_tophat/accepted_hits_noDup_sel_chrX.bam ... All hits are returned with flags.
1 BAM files are combined
*** Only reads from strand - will be considered.
*** Only unique hits are used to compute read count.
*** chr1 do not have any alignment.
*** chr10 do not have any alignment.
*** chr11 do not have any alignment.
*** chr12 do not have any alignment.
*** chr13 do not have any alignment.
*** chr14 do not have any alignment.
*** chr15 do not have any alignment.
*** chr16 do not have any alignment.
*** chr17 do not have any alignment.
*** chr18 do not have any alignment.
*** chr19 do not have any alignment.
*** chr2 do not have any alignment.
*** chr3 do not have any alignment.
*** chr4 do not have any alignment.
*** chr5 do not have any alignment.
*** chr6 do not have any alignment.
*** chr7 do not have any alignment.
*** chr8 do not have any alignment.
*** chr9 do not have any alignment.
*** chrM do not have any alignment.
*** chrY do not have any alignment.

*** 21 pseudoreads are appended to the end.

**B. Run NB HMM on each chromosome

**C. Disambiguate multihits based on posterior

679/3434 multihit reads corresponding to 1250 ambiguous alignments
have been assigned to 679 unique regions with maximum posterior for the enriched state

**D. Re-run NB HMM with unique hits + disambiguated multihits.

*** chr1 do not have any alignment.
*** chr10 do not have any alignment.
*** chr11 do not have any alignment.
*** chr12 do not have any alignment.
*** chr13 do not have any alignment.
*** chr14 do not have any alignment.
*** chr15 do not have any alignment.
*** chr16 do not have any alignment.
*** chr17 do not have any alignment.
*** chr18 do not have any alignment.
*** chr19 do not have any alignment.
*** chr2 do not have any alignment.
*** chr3 do not have any alignment.
*** chr4 do not have any alignment.
*** chr5 do not have any alignment.
*** chr6 do not have any alignment.
*** chr7 do not have any alignment.
*** chr8 do not have any alignment.
*** chr9 do not have any alignment.
*** chrM do not have any alignment.
*** chrY do not have any alignment.

*** 21 pseudoreads are appended to the end.

*III. Seek RIP regions with control library:


*IV. Annotate RIP regions via online ensembl database (mmusculus_gene_ensembl):

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Opening and ending tag mismatch: div line 20 and body
Opening and ending tag mismatch: div line 19 and html
Premature end of data in tag body line 17
Premature end of data in tag html line 2
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2: Entity 'hellip' not defined
3: Entity 'hellip' not defined
4: Entity 'nbsp' not defined
5: Entity 'raquo' not defined
6: Entity 'hellip' not defined
7: Entity 'hellip' not defined
8: Entity 'hellip' not defined
9: Entity 'hellip' not defined
10: Entity 'hellip' not defined
11: Opening and ending tag mismatch: img line 68 and li
12: Opening and ending tag mismatch: li line 68 and ul
13: Opening and ending tag mismatch: ul line 67 and div
14: Opening and ending tag mismatch: ul line 106 and p
15: expected '>'
16: Opening and ending tag mismatch: p line 103 and div
17: Entity 'copy' not defined
18: AttValue: " or ' expected
19: attributes construct error
20: Couldn't find end of Start Tag input line 256
21: Opening and ending tag mismatch: div line 20 and body
22: Opening and ending tag mismatch: div line 19 and html
23: Premature end of data in tag body line 17
24: Premature end of data in tag html line 2

> names(seekOut.PRC2)
Error: object 'seekOut.PRC2' not found

Session Info:

> sessionInfo()
R version 3.3.2 (2016-10-31)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: macOS Sierra 10.12.3

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] grid      parallel  stats4    stats     graphics  grDevices utils     datasets  methods  
[10] base     

other attached packages:
 [1] ChIPpeakAnno_3.8.9         VennDiagram_1.6.17         futile.logger_1.4.3       
 [4] biomaRt_2.30.0             RIPSeeker_1.14.0           rtracklayer_1.34.2        
 [7] GenomicAlignments_1.10.0   Rsamtools_1.26.1           Biostrings_2.42.1         
[10] XVector_0.14.0             SummarizedExperiment_1.4.0 Biobase_2.34.0            
[13] GenomicRanges_1.26.3       GenomeInfoDb_1.10.3        IRanges_2.8.1             
[16] S4Vectors_0.12.1           BiocGenerics_0.20.0        BiocInstaller_1.24.0      
[19] gridExtra_2.2.1            gplots_3.0.1               readr_1.0.0               
[22] gsalib_2.1                 ggplot2_2.2.1             

loaded via a namespace (and not attached):
 [1] Rcpp_0.12.9                   lattice_0.20-34               GO.db_3.4.0                  
 [4] gtools_3.5.0                  assertthat_0.1                digest_0.6.12                
 [7] mime_0.5                      R6_2.2.0                      plyr_1.8.4                   
[10] futile.options_1.0.0          RSQLite_1.1-2                 httr_1.2.1                   
[13] zlibbioc_1.20.0               GenomicFeatures_1.26.3        lazyeval_0.2.0               
[16] gdata_2.17.0                  Matrix_1.2-8                  labeling_0.3                 
[19] splines_3.3.2                 BiocParallel_1.8.1            AnnotationHub_2.6.4          
[22] RCurl_1.95-4.8                munsell_0.4.3                 shiny_1.0.0                  
[25] httpuv_1.3.3                  multtest_2.30.0               htmltools_0.3.5              
[28] tibble_1.2                    interactiveDisplayBase_1.12.0 idr_1.2                      
[31] matrixStats_0.51.0            XML_3.98-1.5                  MASS_7.3-45                  
[34] bitops_1.0-6                  RBGL_1.50.0                   xtable_1.8-2                 
[37] gtable_0.2.0                  DBI_0.6                       scales_0.4.1                 
[40] graph_1.52.0                  KernSmooth_2.23-15            limma_3.30.13                
[43] seqinr_3.3-3                  lambda.r_1.1.9                ensembldb_1.6.2              
[46] tools_3.3.2                   ade4_1.7-5                    BSgenome_1.42.0              
[49] yaml_2.1.14                   survival_2.41-2               AnnotationDbi_1.36.2         
[52] colorspace_1.3-2              regioneR_1.6.2                caTools_1.17.1               
[55] memoise_1.0.0

RIPSeeker ripSeek RIP • 1.5k views

ADD COMMENT • link 7.1 years ago ofonov ▴ 10

0

Entering edit mode

In case you still haven't solved your issue with RIPSeeker:

You should check how your .bam files are annotated. Depending on how you've aligned and annotated your .fastq files chromosomes are annotated either as "Chr1" or "1", which might explain why it is not recognizing any alignment.

ADD REPLY • link 6.7 years ago yuka.takemon • 0