Hello, I have same problem as reported previously (https://stat.ethz.ch/piper mail/bioconductor/2013-January/050555.html), "Error in .doBasicCount(obj)". How and what to fix ? >count.table <- easyRNASeq( + filenames=c("siCTRL_5314.bam", + "siCTRL_5315.bam", + "siZ9_5310.bam", + "siZ9_5312.bam"), + filesDirectory="/Volumes/Cellar/DNASeq_20121016/02.human/Orig_tophat _mapping", + organism="Hsapiens", + chr.sizes=as.list(seqlengths(Hsapiens)), + readLength=101L, + annotationMethod="rda", + annotationFile="./exon.annotation.biomart.rda", + format="bam", + count="exons") Checking arguments... Fetching annotations... Summarizing counts... Processing siCTRL_5314.bam Error in .doBasicCount(obj) : The genomicAnnotation slot is empty >sessionInfo() R version 2.15.2 (2012-10-26) Platform: x86_64-apple-darwin9.8.0/x86_64 (64-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 [8] base other attached packages: [1] BSgenome.Hsapiens.UCSC.hg19_1.3.19 easyRNASeq_1.4.2 [3] ShortRead_1.16.4 latticeExtra_0.6-24 [5] RColorBrewer_1.0-5 Rsamtools_1.10.2 [7] DESeq_1.10.1 lattice_0.20-14 [9] locfit_1.5-8 BSgenome_1.26.1 [11] GenomicRanges_1.10.7 Biostrings_2.26.3 [13] edgeR_3.0.8 limma_3.14.4 [15] Biobase_2.18.0 genomeIntervals_1.14.0 [17] intervals_0.14.0 IRanges_1.16.6 [19] BiocGenerics_0.4.0 biomaRt_2.14.0 loaded via a namespace (and not attached): [1] annotate_1.36.0 AnnotationDbi_1.20.7 bitops_1.0-4.2 [4] DBI_0.2-5 genefilter_1.40.0 geneplotter_1.36.0 [7] grid_2.15.2 hwriter_1.3 RCurl_1.95-4.1 [10] RSQLite_0.11.2 splines_2.15.2 stats4_2.15.2 [13] survival_2.37-4 XML_3.95-0.2 xtable_1.7-1 [16] zlibbioc_1.4.0 >samtools view -H siCTRL_5314.bam @HD VN:1.0 SO:coordinate @SQ SN:chr1 LN:249250621 @SQ SN:chr10 LN:135534747 @SQ SN:chr11 LN:135006516 @SQ SN:chr12 LN:133851895 @SQ SN:chr13 LN:115169878 @SQ SN:chr14 LN:107349540 @SQ SN:chr15 LN:102531392 @SQ SN:chr16 LN:90354753 @SQ SN:chr17 LN:81195210 @SQ SN:chr18 LN:78077248 @SQ SN:chr19 LN:59128983 @SQ SN:chr2 LN:243199373 @SQ SN:chr20 LN:63025520 @SQ SN:chr21 LN:48129895 @SQ SN:chr22 LN:51304566 @SQ SN:chr3 LN:198022430 @SQ SN:chr4 LN:191154276 @SQ SN:chr5 LN:180915260 @SQ SN:chr6 LN:171115067 @SQ SN:chr7 LN:159138663 @SQ SN:chr8 LN:146364022 @SQ SN:chr9 LN:141213431 @SQ SN:chrM LN:16571 @SQ SN:chrX LN:155270560 @SQ SN:chrY LN:59373566 @PG ID:TopHat VN:1.3.2 CL:tophat -p 8 genome siCTRL_5314_R01.txt siCTRL_5314_R02.txt I did not use GTF file for annotation, the ".rda" file was created as follows: ################################################################# library(biomaRt) ensembl<-useMart("ensembl", dataset="hsapiens_gene_ensembl") exon.annotation<-getBM( c("external_gene_id", "ensembl_gene_id", "strand", "ensembl_transcript_id", "chromosome_name", "ensembl_exon_id", "exon_chrom_start", "exon_chrom_end" ), mart=ensembl ) exon.annotation$chromosome <- paste( "chr", exon.annotation$chromosome_name, sep="") library(IRanges) gAnnot <- RangedData( IRanges(start=exon.annotation$exon_chrom_start, end=exon.annotation$exon_chrom_end ), space=exon.annotation$chromosome, strand=exon.annotation$strand, transcript=exon.annotation$ensembl_transcript_id, gene=exon.annotation$ensembl_gene_id, exon=exon.annotation$ensembl_exon_id, gene_name=exon.annotation$external_gene_id, universe="Hsap" ) save(gAnnot, file="exon.annotation.biomart.rda") ################################################################# Thanks in advance. Josef Pasulka CEITEC muni pepap at chemi.muni.cz