[BioC] Getting counts for previously undetected transcripts and genes with easyRNASeq: comparison to Cufflinks
Alex Gutteridge
alexg at ruggedtextile.com
Thu Dec 13 17:12:16 CET 2012
On 13.12.2012 15:55, Richard Friedman wrote:
> On Dec 4, 2012, at 10:54 AM, Richard Friedman wrote:
>
>> On Dec 4, 2012, at 5:27 AM, Nicolas Delhomme wrote:
>>>
>>>
>>> As I said, I have a very similar setup, but completely de-novo.
>>> I've been (still am) testing several approaches:
>>>
>>> 1) running TopHat/Cufflinks/Cuffmerge (cuffmerge to get the
>>> exon/gene GFF) and from that I go back to the original alignments by
>>> tophat and use these as input together with the GFF for easyRNASeq. I
>>> then get my DESeq/edgeR output and proceed in R.
>>
>
>
>
>
> Dear Nico and list,
>
> I tried using the cuffmerge gtf file as the gff file in TopHat as
> you suggest and successfully
> generated a bam file but easyRNASeq could not read it. (Although
> TopHat/Cufflinks are not bioconductor
> programs, it is desirable that their output should be readable by
> bioconductor programs so that
> I hope that including a TopHat command does not go beyond the scope
> of this list).
You'll kick yourself, but it doesn't look like you put quotes around
the file name?
>
> To realign the reads using the gtf file generated by cufflinks I used
>
> tophat -p 6 -G
> /Documents/clients/phyllis/humanlearndata/merged_asm/merged.gtf -o
> SRR490225_it_thout genome SRR490225.fastq
>
> where merged,gtf is the gtf file generated by cufflinks.
>
> Thene here is a record of my easyRNASeq session;
>
>
> ##############################################################################################
>
> R version 2.15.2 (2012-10-26) -- "Trick or Treat"
> Copyright (C) 2012 The R Foundation for Statistical Computing
> ISBN 3-900051-07-0
> Platform: i386-apple-darwin9.8.0/i386 (32-bit)
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> [R.app GUI 1.53 (6335) i386-apple-darwin9.8.0]
>
> [Workspace restored from /Users/friedman/.RData]
> [History restored from /Users/friedman/.Rapp.history]
>
>> library(easyRNASeq)
> Loading required package: parallel
> Loading required package: genomeIntervals
> Loading required package: intervals
> Loading required package: BiocGenerics
>
> Attaching package: ‘BiocGenerics’
>
> The following object(s) are masked from ‘package:stats’:
>
> xtabs
>
> The following object(s) are masked from ‘package:base’:
>
> anyDuplicated, cbind, colnames, duplicated, eval, Filter, Find,
> get, intersect,
> lapply, Map, mapply, mget, order, paste, pmax, pmax.int, pmin,
> pmin.int, Position,
> rbind, Reduce, rep.int, rownames, sapply, setdiff, table, tapply,
> union, unique
>
> Loading required package: Biobase
> Welcome to Bioconductor
>
> Vignettes contain introductory material; view with
> 'browseVignettes()'. To cite
> Bioconductor, see 'citation("Biobase")', and for packages
> 'citation("pkgname")'.
>
> Loading required package: biomaRt
> Loading required package: edgeR
> Loading required package: limma
> Loading required package: Biostrings
> Loading required package: IRanges
>
> Attaching package: ‘IRanges’
>
> The following object(s) are masked from ‘package:intervals’:
>
> expand, reduce
>
>
> Attaching package: ‘Biostrings’
>
> The following object(s) are masked from ‘package:intervals’:
>
> type
>
> Loading required package: BSgenome
> Loading required package: GenomicRanges
> Loading required package: DESeq
> Loading required package: locfit
> locfit 1.5-8 2012-04-25
>
> Attaching package: ‘locfit’
>
> The following object(s) are masked from ‘package:GenomicRanges’:
>
> left, right
>
> Loading required package: lattice
>
> Attaching package: ‘DESeq’
>
> The following object(s) are masked from ‘package:limma’:
>
> plotMA
>
> Loading required package: Rsamtools
> Loading required package: ShortRead
> Loading required package: latticeExtra
> Loading required package: RColorBrewer
> Warning messages:
> 1: replacing previous import ‘coerce’ when loading ‘intervals’
> 2: replacing previous import ‘initialize’ when loading ‘intervals’
>> library(BSgenome.Hsapiens.UCSC.hg19)
>> library(Rsamtools)
>>
>> chr.sizes=seqlengths(Hsapiens)
>> chr.sizes
>
> chr1 chr2 chr3
> chr4
> 249250621 243199373 198022430
> 191154276
>
> ……..
>
>
>> bamfiles=dir(getwd(),pattern="*\\.bam$")
>> bamfiles
> [1] "SRR490224it.bam" "SRR490225it.bam"
>
>> indexBam(SRR490224it.bam)
> Error in indexBam(SRR490224it.bam) :
> error in evaluating the argument 'files' in selecting a method for
> function 'indexBam': Error: object 'SRR490224it.bam' not found
>
>> sessionInfo()
> R version 2.15.2 (2012-10-26)
> Platform: i386-apple-darwin9.8.0/i386 (32-bit)
>
> 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] parallel stats graphics grDevices utils datasets
> methods base
>
> other attached packages:
> [1] BSgenome.Hsapiens.UCSC.hg19_1.3.19 easyRNASeq_1.4.2
> [3] ShortRead_1.16.2 latticeExtra_0.6-19
> [5] RColorBrewer_1.0-5 Rsamtools_1.10.2
> [7] DESeq_1.10.1 lattice_0.20-10
> [9] locfit_1.5-8 BSgenome_1.26.1
> [11] GenomicRanges_1.10.5 Biostrings_2.26.2
> [13] IRanges_1.16.4 edgeR_3.0.4
> [15] limma_3.12.1 biomaRt_2.14.0
> [17] Biobase_2.18.0 genomeIntervals_1.14.0
> [19] BiocGenerics_0.4.0 intervals_0.13.3
>
> loaded via a namespace (and not attached):
> [1] annotate_1.34.1 AnnotationDbi_1.20.2 bitops_1.0-4.1
> DBI_0.2-5
> [5] genefilter_1.38.0 geneplotter_1.34.0 grid_2.15.2
> hwriter_1.3
> [9] RCurl_1.91-1 RSQLite_0.11.1 splines_2.15.2
> stats4_2.15.2
> [13] survival_2.36-14 XML_3.9-4 xtable_1.7-0
> zlibbioc_1.2.0
>
>
>> ls()
> [1] "bamfiles" "chr.sizes"
>
>
> ##############################################################################################
>
> Any suggestions would be appreciated.
>
> Thanks and best wishes,
> Rich
> Richard A. Friedman, PhD
> Associate Research Scientist,
> Biomedical Informatics Shared Resource
> Herbert Irving Comprehensive Cancer Center (HICCC)
> Lecturer,
> Department of Biomedical Informatics (DBMI)
> Educational Coordinator,
> Center for Computational Biology and Bioinformatics (C2B2)/
> National Center for Multiscale Analysis of Genomic Networks (MAGNet)/
> Columbia Initiative in Systems Biology
> Room 824
> Irving Cancer Research Center
> Columbia University
> 1130 St. Nicholas Ave
> New York, NY 10032
> (212)851-4765 (voice)
> friedman at cancercenter.columbia.edu
> http://cancercenter.columbia.edu/~friedman/
>
> In memoriam, Ray Bradbury
>
>
>
>
>
>
>
> [[alternative HTML version deleted]]
--
Alex Gutteridge
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