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Old 04-11-2016, 03:39 PM   #1
Rammohan
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Location: Toronto

Join Date: Dec 2015
Posts: 10
Default BAM files from RNAseq -Alignment (Basespace) not working with DESEq2 in R

Hello experts,

With my recent run in illumina Hiseq2500 I aligned the FASTAQ files generated in basespace to human reference genome (Homosapieans (PAR masked)/hg19 (Refseq) using the RNA-seq Alignment App.I used Tophat (Bowtie2) aligner for the same. except for "NO" to the stranded information, No other parameters were selected.
I downloaded the BAM files generated and used them in the Bioconductor RNAseq workflow (Using DESeq2)
http://www.bioconductor.org/help/workflows/rnaseqGene/

to create a summarized object I used "Homo_sapiens.GRCh37.75.gtf"

below is the script:

(txdb <- makeTxDbFromGFF("Homo_sapiens.GRCh37.75.gtf", format="gtf", circ_seqs=character()))
(ebg <- exonsBy(txdb, by="gene"))

se <- summarizeOverlaps(features=ebg,reads=bamfiles,mode="Union",singleEnd=FALSE,ignore.strand=TRUE,fragments=TRUE )

I am getting the following error message and am unable to get any count

Warning message:
In .Seqinfo.mergexy(x, y) :
The 2 combined objects have no sequence levels in common. (Use
suppressWarnings() to suppress this warning.)

Any suggestions?

Ram
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