Hello, I'm trying to use featureCounts to extract read counts per transcript from the SAM file (generated using Bowtie2) mapped to de novo assembled transcripts (for DE analysis). I made annotation file as per the SAF requirements: Eg: *GeneID Chr Start End Strand* Locus_47_Transcript_16/31_Confidence_0.158_Length_1485 Locus_47_Transcript_16/31_Confidence_0.158_Length_1485 1 1485 + Locus_58_Transcript_85/85_Confidence_0.017_Length_650 Locus_58_Transcript_85/85_Confidence_0.017_Length_650 1 650 + Here I made both the GeneID and the Chr columns same with *Start* being 1 and *End* as the length of the transcript and the Strand as + for all transcripts. When I used the following code, it does nothing for a while and then R crashes. library(Rsubread) counts <- featureCounts(files="AssembledTranscriptome-LMcontrol1.sam",annot="Ann otRsubreadSAF.txt",isGTFAnnotationFile=FALSE) Rsubread works great with GTF as annot parameter on genome mapped SAM files but not with the SAF, as I tried here. Please let me know where I'm wrong here or if anyone tried other packages that serves the purpose. sessionInfo() R version 3.0.1 (2013-05-16) Platform: x86_64-redhat-linux-gnu (64-bit) locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C 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 LC_PAPER=C LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] splines parallel grid stats graphics grDevices utils datasets methods base other attached packages: [1] Rsamtools_1.12.3 edgeR_3.2.4 Rsubread_1.10.5 [4] ChIPpeakAnno_2.8.0 GenomicFeatures_1.12.3 limma_3.16.7 [7] org.Hs.eg.db_2.9.0 GO.db_2.9.0 RSQLite_0.11.4 [10] DBI_0.2-7 AnnotationDbi_1.22.6 BSgenome.Ecoli.NCBI.20080805_1.3.17 [13] BSgenome_1.28.0 GenomicRanges_1.12.4 Biostrings_2.28.0 [16] IRanges_1.18.2 multtest_2.16.0 Biobase_2.20.1 [19] biomaRt_2.16.0 BiocGenerics_0.6.0 VennDiagram_1.6.4 [22] sendmailR_1.1-2 base64enc_0.1-1 BiocInstaller_1.10.3 loaded via a namespace (and not attached): [1] bitops_1.0-5 MASS_7.3-26 RCurl_1.95-4.1 rtracklayer_1.20.4 stats4_3.0.1 [6] survival_2.37-4 tools_3.0.1 XML_3.98-1.1 zlibbioc_1.6.0 [[alternative HTML version deleted]]