I am interested in analysing the differential expression of the transcriptome of A.flavus grown at two different condition using easyRNASeq and DEGSeq. I have the raw reads in fastq format for the expt. I had mapped these reads to the reference genome sequence using bowtie and the output bam file was given as input to easyRNASeq. It gave the error ".doBasicCount(obj) : The genomicAnnotation slot is empty". When I gave the tophat generated bam file (along with the bai), I get the same error. How to overcome this problem. -- output of sessionInfo(): > easyRNASeq(filesDirectory=("/home/Dharmalingam/Documents/Bioconducto r/eg/Aflavus/rnaseq/"), organism="Aflavus", readLength=100L, annotationMethod="gtf", annotationFile="Aspergillus_flavus.JCVI- afl1-v2.0.16 (1).gtf", count="exons", filenames=c("30accepted_hits.bam", "37accepted_hits.bam")) Checking arguments... Fetching annotations... Read 103401 records Summarizing counts... Processing 30accepted_hits.bam Error in .doBasicCount(obj) : The genomicAnnotation slot is empty In addition: Warning messages: 1: In easyRNASeq(filesDirectory = ("/home/Dharmalingam/Documents/Bioconductor/eg/Aflavus/rnaseq/"), : Your organism has no mapping defined to perform the validity check for the UCSC compliance of the chromosome name. Defined organism's mapping can be listed using the 'knownOrganisms' function. To benefit from the validity check, you can provide a 'chr.map' to your 'easyRNASeq' function call. As you did not do so, 'validity.check' is turned off 2: In .Method(..., deparse.level = deparse.level) : number of columns of result is not a multiple of vector length (arg 256) 3: In easyRNASeq(filesDirectory = ("/home/Dharmalingam/Documents/Bioconductor/eg/Aflavus/rnaseq/"), : There are 59 features/exons defined in your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want? -- Sent via the guest posting facility at bioconductor.org.

Hi Jeya, I'm sorry if that's not your name, I just guessed from you email address. Can you post the output of the sessionInfo() command? It appears to be missing from your post. It's a surprising error and it may have to do with your annotation gtf. Could you send me the gtf file or at least an excerpt of it (directly to me off the list)? If you could as well post the header of your bam file (i.e. the results of running samtools view -H /home/Dha rmalingam/Documents/Bioconductor/eg/Aflavus/rnaseq/30accepted_hits.bam ), that would be very useful. Thanks, Nico --------------------------------------------------------------- Nicolas Delhomme Genome Biology Computational Support European Molecular Biology Laboratory Tel: +49 6221 387 8310 Email: nicolas.delhomme at embl.de Meyerhofstrasse 1 - Postfach 10.2209 69102 Heidelberg, Germany --------------------------------------------------------------- On 29 Jan 2013, at 11:53, Jeya [guest] wrote: > > I am interested in analysing the differential expression of the transcriptome of A.flavus grown at two different condition using easyRNASeq and DEGSeq. I have the raw reads in fastq format for the expt. I had mapped these reads to the reference genome sequence using bowtie and the output bam file was given as input to easyRNASeq. It gave the error ".doBasicCount(obj) : The genomicAnnotation slot is empty". When I gave the tophat generated bam file (along with the bai), I get the same error. How to overcome this problem. > > -- output of sessionInfo(): > >> easyRNASeq(filesDirectory=("/home/Dharmalingam/Documents/Bioconduct or/eg/Aflavus/rnaseq/"), organism="Aflavus", readLength=100L, annotationMethod="gtf", annotationFile="Aspergillus_flavus.JCVI- afl1-v2.0.16 (1).gtf", count="exons", filenames=c("30accepted_hits.bam", "37accepted_hits.bam")) > Checking arguments... > Fetching annotations... > Read 103401 records > Summarizing counts... > Processing 30accepted_hits.bam > Error in .doBasicCount(obj) : The genomicAnnotation slot is empty > In addition: Warning messages: > 1: In easyRNASeq(filesDirectory = ("/home/Dharmalingam/Documents/Bioconductor/eg/Aflavus/rnaseq/"), : > Your organism has no mapping defined to perform the validity check for the UCSC compliance of the chromosome name. > Defined organism's mapping can be listed using the 'knownOrganisms' function. > To benefit from the validity check, you can provide a 'chr.map' to your 'easyRNASeq' function call. > As you did not do so, 'validity.check' is turned off > 2: In .Method(..., deparse.level = deparse.level) : > number of columns of result is not a multiple of vector length (arg 256) > 3: In easyRNASeq(filesDirectory = ("/home/Dharmalingam/Documents/Bioconductor/eg/Aflavus/rnaseq/"), : > There are 59 features/exons defined in your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want? > > > -- > Sent via the guest posting facility at bioconductor.org.