Hi, I am using easyRNASeq for estimating the counts in an rna-seq alignment to hg19/GRCh37 done using bowtie2. I would like to get the counts "per gene". The code runs successfully and I get an output table, but the number of records in the output table are ~57000: cat count.tsv | wc -l 57774 I am wondering why the number of counts are so much greater than the total number of genes (~30,000). I am getting some warning message, which may be related to this, especially #1 and #4: Warning messages: 1: Consider using 'synthetic transcripts' as described in the section 7.1 of the vignette instead of the count=genes,summarization=geneModels deprecated paradigm. 2: In easyRNASeq(filesDirectory = getwd(), filenames = c("BRPC13-1118_L1.D710_501.sorted.bam", : You enforce UCSC chromosome conventions, however the provided chromosome size list is not compliant. Correcting it. 3: In easyRNASeq(filesDirectory = getwd(), filenames = c("BRPC13-1118_L1.D710_501.sorted.bam", : You enforce UCSC chromosome conventions, however the provided annotation is not compliant. Correcting it. 4: In easyRNASeq(filesDirectory = getwd(), filenames = c("BRPC13-1118_L1.D710_501.sorted.bam", : There are 18950 synthetic exons as determined from your annotation that overlap! This implies that some reads will be counted more than once! Is that really what you want? 5: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 6: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 7: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. 8: In fetchCoverage(rnaSeq, format = format, filename = filename, filter = filter, : You enforce UCSC chromosome conventions, however the provided alignments are not compliant. Correcting it. Code I am running for estimating the counts: > count.table <- easyRNASeq(filesDirectory=getwd(), + filenames=c("A.sorted.bam","B.sorted.bam","C.sorted.bam","D.sorted.b am"), + organism="Hsapiens", + annotationMethod="gtf", + annotationFile="/general/NGS/index/human/Homo_sapiens.GRCh37.74.gtf", + count="genes", + summarization="geneModels") -- output of sessionInfo(): > sessionInfo() R version 3.0.3 (2014-03-06) Platform: x86_64-unknown-linux-gnu (64-bit) locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=en_US.UTF-8 LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base > > packageDescription("easyRNASeq") Package: easyRNASeq Version: 1.8.7 Date: 2014-03-25 Type: Package Title: Count summarization and normalization for RNA-Seq data. Author: Nicolas Delhomme, Ismael Padioleau, Bastian Schiffthaler Maintainer: Nicolas Delhomme <delhomme at="" embl.de=""> Description: Calculates the coverage of high-throughput short-reads against a genome of reference and summarizes it per feature of interest (e.g. exon, gene, transcript). The data can be normalized as 'RPKM' or by the 'DESeq' or 'edgeR' package. Depends: genomeIntervals (>= 1.18.0), Biobase (>= 2.22.0), biomaRt (>= 2.18.0), edgeR (>= 3.4.0), Biostrings (>= 2.30.0), DESeq (>= 1.14.0), GenomicRanges (>= 1.14.3), IRanges (>= 1.20.5), Rsamtools (>= 1.14.1), ShortRead (>= 1.20.0) Imports: graphics, methods, parallel, utils, BiocGenerics (>= 0.8.0), LSD (>= 2.5) Suggests: BSgenome (>= 1.30.0), BSgenome.Dmelanogaster.UCSC.dm3 (>= 1.3.19), GenomicFeatures (>= 1.14.0), RnaSeqTutorial (>= 0.0.13), BiocStyle (>= 1.0.0) License: Artistic-2.0 LazyLoad: yes biocViews: GeneExpression, RNAseq, Genetics, Preprocessing Packaged: 2014-03-26 04:53:07 UTC; biocbuild Built: R 3.0.3; ; 2014-03-31 20:30:18 UTC; unix -- Sent via the guest posting facility at bioconductor.org.