Hi,

Apparently I need to use a gtf or gff3 file for annotation with easyRNAseq now when processing hg19 bamfiles now - as method biomaRt uses GRCh39 and I get an error. So far so bad.

However, I seem not to be able to download a gtf or gff3 file for hg19 that easyRNAseq would accept:

> DGE<-simpleRNASeq(bamFiles=getBamFileList(bamfiles), param=param, nnodes=4, verbose=T, override=T)

...

==========================
Processing the annotation
==========================
Validating the annotation source
Read 993 records
Error in .validate(obj, verbose = verbose) :
The provided gff3 contains no annotation of type 'mRNA' and/or 'exon' in the first 1000 lines.

I've tried files the following annotation files but none seems to work:

ref_GRCh37.p13_top_level.gff3.gz
ref_GRCh37.p13_scaffolds.gff3.gz
gencode.v19.annotation.gtf
gencode.v19.annotation.gff3
Homo_sapiens.GRCh37.70.gtf

will try the Illumina iGenome next. What am I missing here?!?

Hey Nico,

sorry for the late reply (am currently battling kallisto/sleuth):

> sessionInfo()
R version 3.2.1 (2015-06-18)
Platform: x86_64-unknown-linux-gnu (64-bit)
Running under: Red Hat Enterprise Linux Server release 6.6 (Santiago)

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] stats4    parallel  stats     graphics  grDevices utils     datasets
[8] methods   base

other attached packages:
 [1] DESeq2_1.8.1                      RcppArmadillo_0.5.500.2.0
 [3] Rcpp_0.12.1                       easyRNASeq_2.4.7
 [5] BSgenome.Hsapiens.UCSC.hg19_1.4.0 BSgenome_1.36.3
 [7] rtracklayer_1.28.10               Biostrings_2.36.4
 [9] XVector_0.8.0                     GenomicRanges_1.20.6
[11] GenomeInfoDb_1.4.2                IRanges_2.2.7
[13] S4Vectors_0.6.5                   BiocGenerics_0.14.0

loaded via a namespace (and not attached):
 [1] genefilter_1.50.0       locfit_1.5-9.1          reshape2_1.4.1
 [4] splines_3.2.1           lattice_0.20-33         colorspace_1.2-6
 [7] survival_2.38-3         XML_3.98-1.3            foreign_0.8-66
[10] DBI_0.3.1               BiocParallel_1.2.21     RColorBrewer_1.1-2
[13] lambda.r_1.1.7          plyr_1.8.3              stringr_1.0.0
[16] zlibbioc_1.14.0         munsell_0.4.2           gtable_0.1.2
[19] futile.logger_1.4.1     hwriter_1.3.2           latticeExtra_0.6-26
[22] Biobase_2.28.0          geneplotter_1.46.0      biomaRt_2.24.0
[25] AnnotationDbi_1.30.1    proto_0.3-10            acepack_1.3-3.3
[28] xtable_1.7-4            edgeR_3.10.2            scales_0.3.0
[31] limma_3.24.15           Hmisc_3.16-0            annotate_1.46.1
[34] ShortRead_1.26.0        gridExtra_2.0.0         Rsamtools_1.20.4
[37] ggplot2_1.0.1           digest_0.6.8            stringi_0.5-5
[40] DESeq_1.20.0            grid_3.2.1              tools_3.2.1
[43] bitops_1.0-6            magrittr_1.5            RCurl_1.95-4.7
[46] LSD_3.0                 RSQLite_1.0.0           cluster_2.0.3
[49] Formula_1.2-1           futile.options_1.0.0    MASS_7.3-44
[52] rpart_4.1-10            nnet_7.3-11             GenomicAlignments_1.4.1
[55] genomeIntervals_1.24.1  intervals_0.15.1

I've downloaded the mentioned annotations from:

ftp://ftp.ncbi.nlm.nih.gov/genomes/Homo_sapiens/ARCHIVE/ANNOTATION_RELEASE.105/GFF/

http://www.gencodegenes.org/releases/19.html

http://ftp.ensembl.org/pub/release-70/gtf/homo_sapiens/

Thanks a bunch

Same problem with iGenome:

Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-2014-05-23-16-03-55/Genes/genes.gtf

==========================
Processing the annotation
==========================
Validating the annotation source
Read 993 records
Error in .validate(obj, verbose = verbose) :
  The provided gff3 contains no annotation of type 'mRNA' and/or 'exon' in the first 1000 lines.

...

Might this be the problem:

> annotParam<-AnnotParam(datasource=system.file('extdata','~/Homo_sapiens/Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-2014-05-23-16-03-55/Genes/genes.gtf'))

> annotParam
AnnotParamCharacter object set to retrieve 'gff3' formatted annotation from:

>

?