Hi Nico,
thanks for your quick answer.
Here it is the information you ask, it might be quite lengthy.
Unfortunately I get the same error in generating the RNAseq object as well.

> countDataSet <- easyRNASeq(filesDirectory=getwd(),
+                            organism="HSapiens",
+                            chr.sizes=as.list(seqlengths(Hsapiens)),
+                            readLength=100L,
+                            annotationMethod="biomaRt",
+                            format="bam",
+                            count="exons",
+                            filenames=bamfiles,
+                            normalize=FALSE,
+                            outputFormat="RNAseq",
+                           fitType="local"
+                           )
Checking arguments...
Fetching annotations...
Summarizing counts...
Processing SRR349689_1.fastq.novo.new.chr1.bam
Error in FUN(c("GL000229.1", "GL000200.1", "GL000228.1", "GL000241.1",  :
  (list) object cannot be coerced to type 'integer'
In addition: There were 50 or more warnings (use warnings() to see the first 50)

I also tried using a local annotation file, generated this way

hg19.tx <- makeTranscriptDbFromUCSC(
 genome="hg19",
 tablename="refGene")
exon.range<-exons(hg19.tx)
gAnnot <- RangedData(exon.range)
colnames(gAnnot)[2]<-"exon"
save(gAnnot,file="gAnnot.rda")

but then the error is the same.

> countDataSet <- easyRNASeq(filesDirectory=getwd(),
+                            organism="HSapiens",
+                            chr.sizes=as.list(seqlengths(Hsapiens)),
+                            readLength=100L,
+                            annotationMethod="rda",
+                            annotationFile="gAnnot.rda",
+                            format="bam",
+                            count="exons",
+                            filenames=bamfiles,
+                            normalize=TRUE,
+                            outputFormat="DESeq",
+                            conditions=conditions,
+                           fitType="local"
+                           )
Checking arguments...
Fetching annotations...
Summarizing counts...
Processing SRR349689_1.fastq.novo.new.chr1.bam
Error in FUN(c("GL000229.1", "GL000200.1", "GL000228.1", "GL000241.1",  :
  (list) object cannot be coerced to type 'integer'


The .bam file has been generated following Novocraft instructions, from a combined reference of the genome, the transcriptome and the junctions and then recomposed with USeq.
(http://www.novocraft.com/wiki/tiki-index.php?page=RNASeq+analysis%3A+mRNA+and+the+Spliceosome&structure=Novocraft+Technologies&page_ref_id=35)

is there any other information/file that could help in tracking the issue?

thanks,
Francesco



---bam header---
samtools view -H SRR349689_1.fastq.novo.new.chr1.bam
@HD VN:1.0 SO:unsorted
@PG ID:SamTranscriptomeParser CL: args -f SRR349689_1.fastq.novo.sam -u -a 100000 -n 100000 -s SRR349689_1.fastq.novo_converted.sam
@RG ID:unknownReadGroup SM:unknownSample
@SQ SN:GL000229.1 AS:trascriptome.nix LN:119339
@SQ SN:GL000200.1 AS:trascriptome.nix LN:277914
@SQ SN:GL000228.1 AS:trascriptome.nix LN:228349
@SQ SN:GL000241.1 AS:trascriptome.nix LN:136814
@SQ SN:GL000219.1 AS:trascriptome.nix LN:220315
@SQ SN:GL000191.1 AS:trascriptome.nix LN:202431
@SQ SN:GL000242.1 AS:trascriptome.nix LN:142826
@SQ SN:GL000243.1 AS:trascriptome.nix LN:106738
@SQ SN:22 AS:trascriptome.nix LN:51342124
@SQ SN:GL000245.1 AS:trascriptome.nix LN:132396
@SQ SN:MT AS:trascriptome.nix LN:116551
@SQ SN:3 AS:trascriptome.nix LN:198058734
@SQ SN:GL000217.1 AS:trascriptome.nix LN:224716
@SQ SN:2 AS:trascriptome.nix LN:243284564
@SQ SN:1 AS:trascriptome.nix LN:249336112
@SQ SN:7 AS:trascriptome.nix LN:159196479
@SQ SN:6 AS:trascriptome.nix LN:171154887
@SQ SN:GL000215.1 AS:trascriptome.nix LN:232888
@SQ SN:5 AS:trascriptome.nix LN:181002586
@SQ SN:4 AS:trascriptome.nix LN:191137854
@SQ SN:9 AS:trascriptome.nix LN:141252793
@SQ SN:GL000244.1 AS:trascriptome.nix LN:129122
@SQ SN:GL000216.1 AS:trascriptome.nix LN:230528
@SQ SN:8 AS:trascriptome.nix LN:146387700
@SQ SN:GL000218.1 AS:trascriptome.nix LN:197377
@SQ SN:GL000248.1 AS:trascriptome.nix LN:124693
@SQ SN:GL000224.1 AS:trascriptome.nix LN:226253
@SQ SN:GL000210.1 AS:trascriptome.nix LN:123989
@SQ SN:19 AS:trascriptome.nix LN:59216288
@SQ SN:17 AS:trascriptome.nix LN:81293300
@SQ SN:18 AS:trascriptome.nix LN:78015394
@SQ SN:GL000194.1 AS:trascriptome.nix LN:209503
@SQ SN:15 AS:trascriptome.nix LN:102616513
@SQ SN:16 AS:trascriptome.nix LN:90388655
@SQ SN:13 AS:trascriptome.nix LN:115207096
@SQ SN:14 AS:trascriptome.nix LN:107386086
@SQ SN:GL000195.1 AS:trascriptome.nix LN:278899
@SQ SN:11 AS:trascriptome.nix LN:134990552
@SQ SN:12 AS:trascriptome.nix LN:133927113
@SQ SN:GL000225.1 AS:trascriptome.nix LN:273676
@SQ SN:21 AS:trascriptome.nix LN:48217233
@SQ SN:20 AS:trascriptome.nix LN:63026254
@SQ SN:GL000193.1 AS:trascriptome.nix LN:214851
@SQ SN:GL000246.1 AS:trascriptome.nix LN:126826
@SQ SN:GL000237.1 AS:trascriptome.nix LN:117774
@SQ SN:GL000204.1 AS:trascriptome.nix LN:180839
@SQ SN:Y AS:trascriptome.nix LN:59133001
@SQ SN:GL000247.1 AS:trascriptome.nix LN:135428
@SQ SN:X AS:trascriptome.nix LN:155357811
@SQ SN:GL000205.1 AS:trascriptome.nix LN:219192
@SQ SN:GL000192.1 AS:trascriptome.nix LN:644506
@SQ SN:GL000227.1 AS:trascriptome.nix LN:203665
@SQ SN:GL000197.1 AS:trascriptome.nix LN:132711
@SQ SN:GL000236.1 AS:trascriptome.nix LN:126779
@SQ SN:GL000211.1 AS:trascriptome.nix LN:262752
@SQ SN:GL000240.1 AS:trascriptome.nix LN:139915
@SQ SN:GL000239.1 AS:trascriptome.nix LN:123498
@SQ SN:GL000232.1 AS:trascriptome.nix LN:102363
@SQ SN:GL000212.1 AS:trascriptome.nix LN:282254
@SQ SN:GL000238.1 AS:trascriptome.nix LN:132164
@SQ SN:GL000233.1 AS:trascriptome.nix LN:108194
@SQ SN:GL000249.1 AS:trascriptome.nix LN:137299
@SQ SN:GL000223.1 AS:trascriptome.nix LN:280265
@SQ SN:GL000199.1 AS:trascriptome.nix LN:241559
@SQ SN:10 AS:trascriptome.nix LN:135619864
@SQ SN:GL000209.1 AS:trascriptome.nix LN:167344
@SQ SN:GL000196.1 AS:trascriptome.nix LN:132047
@SQ SN:GL000202.1 AS:trascriptome.nix LN:45488
@SQ SN:GL000220.1 AS:trascriptome.nix LN:261756
@SQ SN:GL000214.1 AS:trascriptome.nix LN:224473
@SQ SN:GL000198.1 AS:trascriptome.nix LN:91607
@SQ SN:GL000234.1 AS:trascriptome.nix LN:32013
@SQ SN:GL000213.1 AS:trascriptome.nix LN:237085
@SQ SN:GL000221.1 AS:trascriptome.nix LN:182753
@SQ SN:GL000222.1 AS:trascriptome.nix LN:264765
@SQ SN:GL000206.1 AS:trascriptome.nix LN:138000


----------lengths-----------------
$chr1
[1] 249250621

$chr2
[1] 243199373

$chr3
[1] 198022430

$chr4
[1] 191154276

$chr5
[1] 180915260

$chr6
[1] 171115067

$chr7
[1] 159138663

$chr8
[1] 146364022

$chr9
[1] 141213431

$chr10
[1] 135534747

$chr11
[1] 135006516

$chr12
[1] 133851895

$chr13
[1] 115169878

$chr14
[1] 107349540

$chr15
[1] 102531392

$chr16
[1] 90354753

$chr17
[1] 81195210

$chr18
[1] 78077248

$chr19
[1] 59128983

$chr20
[1] 63025520

$chr21
[1] 48129895

$chr22
[1] 51304566

$chrX
[1] 155270560

$chrY
[1] 59373566

$chrM
[1] 16571

$chr1_gl000191_random
[1] 106433

$chr1_gl000192_random
[1] 547496

$chr4_ctg9_hap1
[1] 590426

$chr4_gl000193_random
[1] 189789

$chr4_gl000194_random
[1] 191469

$chr6_apd_hap1
[1] 4622290

$chr6_cox_hap2
[1] 4795371

$chr6_dbb_hap3
[1] 4610396

$chr6_mann_hap4
[1] 4683263

$chr6_mcf_hap5
[1] 4833398

$chr6_qbl_hap6
[1] 4611984

$chr6_ssto_hap7
[1] 4928567

$chr7_gl000195_random
[1] 182896

$chr8_gl000196_random
[1] 38914

$chr8_gl000197_random
[1] 37175

$chr9_gl000198_random
[1] 90085

$chr9_gl000199_random
[1] 169874

$chr9_gl000200_random
[1] 187035

$chr9_gl000201_random
[1] 36148

$chr11_gl000202_random
[1] 40103

$chr17_ctg5_hap1
[1] 1680828

$chr17_gl000203_random
[1] 37498

$chr17_gl000204_random
[1] 81310

$chr17_gl000205_random
[1] 174588

$chr17_gl000206_random
[1] 41001

$chr18_gl000207_random
[1] 4262

$chr19_gl000208_random
[1] 92689

$chr19_gl000209_random
[1] 159169

$chr21_gl000210_random
[1] 27682

$chrUn_gl000211
[1] 166566

$chrUn_gl000212
[1] 186858

$chrUn_gl000213
[1] 164239

$chrUn_gl000214
[1] 137718

$chrUn_gl000215
[1] 172545

$chrUn_gl000216
[1] 172294

$chrUn_gl000217
[1] 172149

$chrUn_gl000218
[1] 161147

$chrUn_gl000219
[1] 179198

$chrUn_gl000220
[1] 161802

$chrUn_gl000221
[1] 155397

$chrUn_gl000222
[1] 186861

$chrUn_gl000223
[1] 180455

$chrUn_gl000224
[1] 179693

$chrUn_gl000225
[1] 211173

$chrUn_gl000226
[1] 15008

$chrUn_gl000227
[1] 128374

$chrUn_gl000228
[1] 129120

$chrUn_gl000229
[1] 19913

$chrUn_gl000230
[1] 43691

$chrUn_gl000231
[1] 27386

$chrUn_gl000232
[1] 40652

$chrUn_gl000233
[1] 45941

$chrUn_gl000234
[1] 40531

$chrUn_gl000235
[1] 34474

$chrUn_gl000236
[1] 41934

$chrUn_gl000237
[1] 45867

$chrUn_gl000238
[1] 39939

$chrUn_gl000239
[1] 33824

$chrUn_gl000240
[1] 41933

$chrUn_gl000241
[1] 42152

$chrUn_gl000242
[1] 43523

$chrUn_gl000243
[1] 43341

$chrUn_gl000244
[1] 39929

$chrUn_gl000245
[1] 36651

$chrUn_gl000246
[1] 38154

$chrUn_gl000247
[1] 36422

$chrUn_gl000248
[1] 39786

$chrUn_gl000249
[1] 38502



On 7 Feb 2012, at 13:00, Nicolas Delhomme wrote:

Hi Francesco,

I'd need more info to help you.

Can you post the header of your bam files and the content of as.list(seqlengths(Hsapiens)).

In the meanwhile can you run easyRNASeq with normalize set to FALSE, no conditions and the outputFormat set to "RNAseq"?

That will give you back the RNAseq object and we can have a look at what is happening in there.

I get the feeling that it has to do with the exon names being converted into integers (through the use of a factor maybe) but I thought I got that bug squashed already...

Cheers,

Nico

---------------------------------------------------------------
Nicolas Delhomme

Genome Biology Computational Support

European Molecular Biology Laboratory

Tel: +49 6221 387 8310
Email: nicolas.delhomme@embl.de<mailto:nicolas.delhomme@embl.de>
Meyerhofstrasse 1 - Postfach 10.2209
69102 Heidelberg, Germany
---------------------------------------------------------------





On 7 Feb 2012, at 13:37, Lescai, Francesco wrote:

Hi there,
I'm preparing a tutorial, therefore I sliced two RNASeq bam files from chromosome one, in order to reduce the file size.

However, when I count the reads as it follows I have an error.
thanks,
Francesco


countDataSet <- easyRNASeq(filesDirectory=getwd(),
+                            organism="HSapiens",
+                            chr.sizes=as.list(seqlengths(Hsapiens)),
+                            readLength=100L,
+                            annotationMethod="biomaRt",
+                            format="bam",
+                            count="exons",
+                            filenames=bamfiles,
+                            normalize=TRUE,
+                            outputFormat="DESeq",
+                            conditions=conditions,
+                           fitType="local"
+                           )
Checking arguments...
Fetching annotations...
Summarizing counts...
Processing SRR349689_1.fastq.novo.new.chr1.bam
Error in FUN(c("GL000229.1", "GL000200.1", "GL000228.1", "GL000241.1",  :
(list) object cannot be coerced to type 'integer'

I also tried selecting the chromosome with chr.sel=c("1") or chr.sel=c("chr1") but in both cases I get the error
Make sure that your file is valid, that your 'chr.sel' (if provided) contains valid values; i.e. values as found in the file, not as returned by 'RNAseq'.

The alignment has been done using the ENSEMBL reference, i.e. with chromosome listed as "1".

sessionInfo()
R Under development (unstable) (2012-01-20 r58146)
Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit)

locale:
[1] C/en_US.UTF-8/C/C/C/C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] GenomicFeatures_1.6.7              AnnotationDbi_1.17.15              easyRNASeq_1.1.5
[4] ShortRead_1.13.13                  latticeExtra_0.6-19                RColorBrewer_1.0-5
[7] Rsamtools_1.7.27                   genomeIntervals_1.11.0             intervals_0.13.3
[10] DESeq_1.7.6                        locfit_1.5-6                       lattice_0.20-0
[13] akima_0.5-7                        BiocInstaller_1.3.7                Biobase_2.15.3
[16] edgeR_2.5.9                        limma_3.11.11                      biomaRt_2.11.1
[19] BSgenome.Hsapiens.UCSC.hg19_1.3.17 BSgenome_1.23.2                    Biostrings_2.23.6
[22] GenomicRanges_1.7.16               IRanges_1.13.22                    BiocGenerics_0.1.4

loaded via a namespace (and not attached):
[1] DBI_0.2-5          RCurl_1.9-5        RSQLite_0.11.1     XML_3.8-0          annotate_1.33.2
[6] bitops_1.0-4.1     genefilter_1.37.0  geneplotter_1.33.1 grid_2.15.0        hwriter_1.3
[11] rtracklayer_1.15.7 splines_2.15.0     survival_2.36-10   tools_2.15.0       xtable_1.6-0
[16] zlibbioc_1.1.1


---------------------------------------------------------------------------------
Francesco Lescai, PhD, EDBT
Senior Research Associate in Genome Analysis
University College London
Faculty of Population Health Sciences
Dept. Genes, Development & Disease
ICH - Molecular Medicine Unit, GOSgene team
30 Guilford Street
WC1N 1EH London UK

email: f.lescai@ucl.ac.uk<mailto:f.lescai@ucl.ac.uk><mailto:f.lescai@ucl.ac.uk>
phone: +44.(0)207.905.2274
[ext: 2274]
--------------------------------------------------------------------------------


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---------------------------------------------------------------------------------
Francesco Lescai, PhD, EDBT
Senior Research Associate in Genome Analysis
University College London
Faculty of Population Health Sciences
Dept. Genes, Development & Disease
ICH - Molecular Medicine Unit, GOSgene team
30 Guilford Street
WC1N 1EH London UK

email: f.lescai@ucl.ac.uk<mailto:f.lescai@ucl.ac.uk>
phone: +44.(0)207.905.2274
[ext: 2274]
--------------------------------------------------------------------------------


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