Dear Yang,

The output of the script is : 59 59
Therefore, it is not an issue with the length of the feature/chromosome
name (which are same in my case).

I greatly appreciate your help,
Alan



On Sat, Aug 3, 2013 at 6:36 PM, Yang Liao <liao@wehi.edu.au> wrote:

> Dear Alan,
>
> Thank you for using Rsubread. The number of transcripts in the annotation
> file would not be an issue -- our featureCounts function supports
> unlimited number of annotations and chromosomes/transcripts (actually, at
> most 2 billions).
>
> I have just tried to run the in-development version of Rsubread to assign
> reads in your last mail, using the two-line annotation in the other mail.
> The program finished with no errors. The version number I tested was
> "Rsubread_1.11.10".
>
> If this is the version you are using, could you please run this command?
>
> $ cat my_annotations.txt | awk 'BEGIN{max_feature=0; max_chro=0}
> length($1)>max_feature{max_feature=length($1) }
> length($2)>max_chro{max_chro=length($2)} END{print max_feature, max_chro}'
>
>
> where "my_annotations.txt" is the annotation file in SAF format. This
> command will give the length of the longest feature name and the longest
> chromosome name. If either of them is longer than 99, it is still too long
> to our program.
>
> Thank you.
>
> Cheers,
>
> Yang
>
> > Dear Wei,
> >
> > I tried with the devel version but still the problem persists (R
> crashed).
> > Does this got to do with the large number of transcripts (129310) in the
> > file?
> >
> > Could you also please comment on whether if the count data obtained from
> > Rsubread processed on de novo assembled transcripts be used for edgeR
> > differential expression analysis. Or using RSEM to obtain read count
> > information is the way to go?
> >
> > Thank you,
> > Alan
> >
> >
> > On Fri, Aug 2, 2013 at 8:35 PM, Wei Shi <shi@wehi.edu.au> wrote:
> >
> >> Dear Alan,
> >>
> >> Thanks for sending through the data which helped us to identify the
> >> problem. The problem was that featureCounts did not allow the chromosome
> >> names in the provided annotation file to be longer than 48 characters.
> >> We
> >> have increased this limit to 100 characters. This solved the problem and
> >> featureCounts worked fine for your data now from our testing.
> >>
> >> We have committed the changes to bioc devel repository and the latest
> >> version should available in a couple of days (Rsubread v1.11.11).
> >>
> >> Note that we have also made some changes to parameters used by
> >> featureCounts. The 'annot' parameter is now changed to 'annot.ext' and
> >> the
> >> 'genome' parameter changed to 'annot.inbuilt'. This makes it more
> >> clearer
> >> on how to use our in-built annotations and how to provide external
> >> annotations.
> >>
> >> Let me know if the problem you encountered persists.
> >>
> >>
> >> Best wishes,
> >>
> >> Wei
> >>
> >> On Aug 2, 2013, at 2:06 PM, Alan Smith wrote:
> >>
> >> Hi Wei,
> >>
> >> Thanks for looking into it. Here are few lines from my SAM file:
> >>
> >> @HD VN:1.0 SO:unsorted
> >> @SQ SN:Locus_1_Transcript_1/43_Confidence_0.175_Length_1072 LN:1072
> >> @SQ SN:Locus_1_Transcript_6/43_Confidence_0.105_Length_219 LN:219
> >> @SQ SN:Locus_1_Transcript_8/43_Confidence_0.088_Length_258 LN:258
> >> @SQ SN:Locus_1_Transcript_10/43_Confidence_0.088_Length_421 LN:421
> >> @SQ SN:Locus_1_Transcript_14/43_Confidence_0.105_Length_217 LN:217
> >> @SQ SN:Locus_1_Transcript_15/43_Confidence_0.105_Length_392 LN:392
> >> @SQ SN:Locus_1_Transcript_20/43_Confidence_0.123_Length_547 LN:547
> >> @SQ SN:Locus_1_Transcript_21/43_Confidence_0.105_Length_408 LN:408
> >> @SQ SN:Locus_1_Transcript_23/43_Confidence_0.070_Length_246 LN:246
> >>
> >> *This goes for 129310 lines (number of assembled transcripts) then at
> >> the end of the file:*
> >>
> >>
> >> HWI-EAS146_0380:5:100:14414:21375#0/1        16
>  Locus_21817_Transcript_4/5_Confidence_0.200_Length_1641 933     0
> 26M     *       0       0       TCTCGGGGCTCCACTGGAGATGGTCC
>  ^Qa]cf]\U\Z_Zbbeccfaf]`][S      AS:i:-12        XN:i:0  XM:i:2  XO:i:0
>  XG:i:0  NM:i:2  MD:Z:0C0A24     YT:Z:UU
> >> HWI-EAS146_0380:5:100:14769:21375#0/1        16
>  Locus_3777_Transcript_1/1_Confidence_0.000_Length_2776  283     42
>  26M     *       0       0       ATCACATCTCTTTACAGAACCCACTT
>  c]fbdbafdaeafdfdffeffa_aa^      AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0
>  NM:i:0  MD:Z:26 YT:Z:UU
> >> HWI-EAS146_0380:5:100:14923:21376#0/1        4       *       0       0
>       *       *       0       0       ACGACGAAGAAGATGACGACGAGGAC
>  ^^\^Y^W[]aa_cW^ddb``a`WcfR      YT:Z:UU
> >> HWI-EAS146_0380:5:100:15383:21374#0/1        4       *       0       0
>       *       *       0       0       CAACCTTCACCGTGGGTTCTAATGTA
>  YY\\_fff[fd^^^]ZZ]^Wc]c]_a      YT:Z:UU
> >> HWI-EAS146_0380:5:100:15873:21375#0/1        4       *       0       0
>       *       *       0       0       CCTGCTTAAAGAGATCGGAAGAGCGG
>  `_Z^acRcccffYf[ca]d]ff[cdf      YT:Z:UU
> >> HWI-EAS146_0380:5:100:16039:21373#0/1        16
>  Locus_4044_Transcript_6/48_Confidence_0.066_Length_596  412     42
>  26M     *       0       0       TGCAAGCATTCCTCCTGTGGAGGGGT
>  hghedhgdhcgceffdhhhhgeeeee      AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0
>  NM:i:0  MD:Z:26 YT:Z:UU
> >> HWI-EAS146_0380:5:100:16121:21377#0/1        16
>  Locus_2303_Transcript_7/11_Confidence_0.500_Length_1785 1287    42
>  26M     *       0       0       GCTCCGTTTGGATCCGAGAATAGCAC
>  fchhhghhfhgghhfgghhhh\a\\\      AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0
>  NM:i:0  MD:Z:26 YT:Z:UU
> >> HWI-EAS146_0380:5:100:16914:21379#0/1        4       *       0       0
>       *       *       0       0       CCATTCATCATCTTGATACTGCAGAT
>  fffffhhghfghhhhhhhhhhhehea      YT:Z:UU
> >> HWI-EAS146_0380:5:100:16964:21378#0/1        0
> Locus_17940_Transcript_2/7_Confidence_0.538_Length_1249 584     42      26M
>     *       0       0       CTGTAGCCTTTCAGTGAGCTGAGAAA
>  `b^``effffgfgfdgegggedgggg      AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0
>  NM:i:0  MD:Z:26 YT:Z:UU
> >> HWI-EAS146_0380:5:100:17356:21373#0/1        4       *       0       0
>       *       *       0       0       GCCATCAAACCTTACCAATGCTGAGA
>  dacccf[afffffffffdfff]ffca      YT:Z:UU
> >> HWI-EAS146_0380:5:100:17415:21379#0/1        4       *       0       0
>       *       *       0       0       CTAGATGCCTCTTCAGGACTTGAGAA
>  a`a`aggggffffffgaggdfcdfdg      YT:Z:UU
> >>
> >>
> >> Thanks again,
> >>
> >> Alan
> >>
> >>
> >>
> >>
> >>
> >> On Thu, Aug 1, 2013 at 10:55 PM, Wei Shi <shi@wehi.edu.au> wrote:
> >>
> >>> Dear Alan,
> >>>
> >>> Could you please provide us a few reads in your SAM file so that we can
> >>> reproduce the problem? Sorry for your crashed R session.
> >>>
> >>> Best wishes,
> >>> Wei
> >>>
> >>> On Aug 2, 2013, at 12:47 PM, Alan Smith wrote:
> >>>
> >>> > 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="AnnotRsubreadSAF.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]]
> >>> >
> >>> > _______________________________________________
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> >>> > Bioconductor@r-project.org
> >>> > https://stat.ethz.ch/mailman/listinfo/bioconductor
> >>> > Search the archives:
> >>> http://news.gmane.org/gmane.science.biology.informatics.conductor
> >>>
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> >>>
> >>
> >>
> >>
> >> ______________________________________________________________________
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