Hello all, I'm trying to use DEXSeq to identify and quantitate alternative splicing events following transgene expression. I've used BWA to map my reads and now have the resulting SAM files. However, when I use dexseq_count (a Python script) to convert this to a ExonCountSet, I get the following error message: "/common/groups/dac/Hawaii/my_new_env/lib/python2.6/site- packages/HTSeq/__init__.py:592: UserWarning: Read HWUSI- EAS381R:1:10:12324:1388#CGATGT claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)" I can see that my SAM file is not "sorted" properly. Does anyone have a workaround for this? I've searched all over and all I can find is people writing, "I used a perl script as a workaround." Thanks in advance, Wyatt -- output of sessionInfo(): R version 2.14.0 (2011-10-31) 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=C 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 other attached packages: [1] DEXSeq_1.0.2 Biobase_2.14.0 loaded via a namespace (and not attached): [1] hwriter_1.3 plyr_1.6 statmod_1.4.14 stringr_0.6 -- Sent via the guest posting facility at bioconductor.org.

you can sort them by linux command sort -T . -S 2G -k 1,1 -k 3,3 $target_fa.psl.sam > $target_fa.psl.nameSorted.sam -- shan gao Room 231(Dr.Fei lab) Boyce Thompson Institute Cornell University Tower Road, Ithaca, NY 14853-1801 Office phone: 1-607-254-1267(day) Official email:sg839 at cornell.edu Facebook:http://www.facebook.com/profile.php?id=100001986532253

Hi Wyatt, I use SAMTOOLS to sort sam files: http://samtools.sourceforge.net/samtools.shtml ________________________________________ From: bioconductor-bounces@r-project.org [bioconductor- bounces@r-project.org] on behalf of Wyatt McMahon [guest] [guest@bioconductor.org] Sent: 13 December 2011 14:42 To: bioconductor at r-project.org; wmcmahon at vbi.vt.edu Subject: [BioC] deqseq_count and BWA-based SAM files Hello all, I'm trying to use DEXSeq to identify and quantitate alternative splicing events following transgene expression. I've used BWA to map my reads and now have the resulting SAM files. However, when I use dexseq_count (a Python script) to convert this to a ExonCountSet, I get the following error message: "/common/groups/dac/Hawaii/my_new_env/lib/python2.6/site- packages/HTSeq/__init__.py:592: UserWarning: Read HWUSI- EAS381R:1:10:12324:1388#CGATGT claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)" I can see that my SAM file is not "sorted" properly. Does anyone have a workaround for this? I've searched all over and all I can find is people writing, "I used a perl script as a workaround." Thanks in advance, Wyatt -- output of sessionInfo(): R version 2.14.0 (2011-10-31) 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=C 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 other attached packages: [1] DEXSeq_1.0.2 Biobase_2.14.0 loaded via a namespace (and not attached): [1] hwriter_1.3 plyr_1.6 statmod_1.4.14 stringr_0.6 -- Sent via the guest posting facility at bioconductor.org. _______________________________________________ Bioconductor mailing list Bioconductor at r-project.org https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor