[Bioc-devel] Gene annotation: TxDb vs ENSEMBL/NCBI inconsistency

Dear Martin,

the AnnotationHub approach looks awesome! However, somehow it does not work for
me, I always get an error:

library(AnnotationHub)
library(Rsamtools)
library(GenomicFeatures)
ah <- AnnotationHub()

## somehow I don't see DNA sequences for release-80... thus using 75
query(ah, c("Takifugu", "release-75"))

## load the gtf and the dna.toplevel.fa
gtf <- ah[["AH10717"]]
dna <- ah[["AH20637"]]

## create txdb and get exons:
txdb <- makeTxDbFromGRanges(gtf)
exons <- exons(txdb)

## getting the sequences
getSeq(dna, exons)

and I get the following error:
Error in value[[3L]](cond) : 'open' index failed
   file: /Users/jo/~/.AnnotationHub/24732
In addition: Warning message:
In doTryCatch(return(expr), name, parentenv, handler) :
   [fai_load] fail to open FASTA index.

## creating the index
indexFa(dna)

## trying again:
getSeq(dna, exons)
Error in value[[3L]](cond) :  record 1 (MT:1-68) was truncated
   file: /Users/jo/~/.AnnotationHub/24732

This one is from the current R-devel, but I get the same error with the stable
version... any idea?

cheers, jo


my session info:

 > sessionInfo()

R Under development (unstable) (2015-06-06 r68485)
Platform: x86_64-apple-darwin14.4.0/x86_64 (64-bit)
Running under: OS X 10.10.4 (Yosemite)

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

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets
[8] methods   base

other attached packages:
  [1] GenomicFeatures_1.20.1 AnnotationDbi_1.30.1   Biobase_2.28.0
  [4] Rsamtools_1.20.4       Biostrings_2.36.1      XVector_0.8.0
  [7] GenomicRanges_1.20.4   GenomeInfoDb_1.4.0     IRanges_2.2.3
[10] S4Vectors_0.6.0        BiocGenerics_0.14.0    AnnotationHub_2.0.2

loaded via a namespace (and not attached):
  [1] Rcpp_0.11.6                  magrittr_1.5
  [3] GenomicAlignments_1.4.1      zlibbioc_1.14.0
  [5] BiocParallel_1.2.2           xtable_1.7-4
  [7] R6_2.0.1                     stringr_1.0.0
  [9] httr_0.6.1                   tools_3.3.0
[11] DBI_0.3.1                    lambda.r_1.1.7
[13] futile.logger_1.4.1          htmltools_0.2.6
[15] digest_0.6.8                 interactiveDisplayBase_1.6.0
[17] shiny_0.12.0                 rtracklayer_1.28.4
[19] futile.options_1.0.0         bitops_1.0-6
[21] biomaRt_2.24.0               RCurl_1.95-4.6
[23] RSQLite_1.0.0                mime_0.3
[25] stringi_0.4-1                BiocInstaller_1.18.2
[27] XML_3.98-1.2                 httpuv_1.3.2

On 09 Jun 2015, at 16:25, Martin Morgan <mtmorgan at fredhutch.org
<mailto:mtmorgan at fredhutch.org>> wrote:

On 06/08/2015 11:43 PM, Rainer Johannes wrote:

dear Robert and Ludwig,

the EnsDb packages provide all the gene/transcript etc annotations for all
genes defined in the Ensembl database (for a given species and Ensembl
release). Except the column/attribute "entrezid" that is stored in the
internal database there is however no link to NCBI or UCSC annotations. So,
basically, if you want to use "pure" Ensembl based annotations: use EnsDb, if
you want to have the UCSC annotations: use the TxDb packages.

In case you need EnsDbs of other species or Ensembl versions, the ensembldb
package provides functionality to generate such packages either using the
Ensembl Perl API or using GTF files provided by Ensembl. If you have problems
building the packages, just drop me a line and I'll do that.

Two other sources of Ensembl TxDb's are GenomicFeatures::makeTxDbFromBiomart()
and AnnotationHub. For the latter, I'll add a variant of the following to the
AnnotationHub HOWTO
vignettehttp://bioconductor.org/packages/devel/bioc/html/AnnotationHub.htmllater
today.

## Gene models

_Bioconductor_ represents gene models using 'transcript'
databases. These are available via packages such as
[TxDb.Hsapiens.UCSC.hg38.knownGene](http://bioconductor.org/packages/TxDb.Hsapiens.UCSC.knownGene.html),
or can be constructed using functions such as
`[GenomicFeatures](http://bioconductor.org/packages/GenomicFeatures.html)::makeTxDbFromBiomart()`
or `GenomicFeatures::makeTxDbFromGRanges()`.

_AnnotationHub_ provides an easy way to work with gene models
published by Ensembl. Here we discover the Ensemble release 80 r
esources for pufferfish,_Takifugu rubripes_

```{r takifugu-gene-models}
query(ah, c("Takifugu", "release-80"))
```

We see that there is a GTF file, as well as various DNA
sequences. Let's retrieve the GTF and top-level sequence files. The
GTF file is imported as a _GRanges_ instance, the DNA sequence as a
compressed, indexed Fasta file


```{r takifugi-data}
gtf <- ah[["AH47101"]]
dna <- ah[["AH47477"]]

head(gtf, 3)
dna
head(seqlevels(dna))
```

It is trivial to make a TxDb instance

```{r takifugi-txdb}
library(GenomicFeatures)
txdb <- makeTxDbFromGRanges(gtf)
````

and to use that in conjunction with the DNA sequence, e.g., to find
exon sequences of all annotated genes.

```{r takifugi-exons}
library(Rsamtools)     # for getSeq,FaFile-method
exons <- exons(txdb)
getSeq(dna, exons)
```

Some difficulties arise when working with this partly assembled genome
that require more advanced GenomicRanges skills, see the
[GenomicRanges](http://bioconductor.org/packages/GenomicRanges.html)
vignettes, especially "GenomicRanges HOWTOs" and "An Introduction to
GenomicRanges".

cheers, jo

On 03 Jun 2015, at 15:56, Robert M. Flight <rflight79 at gmail.com
<mailto:rflight79 at gmail.com>> wrote:

Ludwig,

If you do this search on the UCSC genome browser (which this annotation
package is built from), you will see that the longest variant is what is
shown

http://genome.ucsc.edu/cgi-bin/hgTracks?clade=mammal&org=Human&db=hg38&position=brca1&hgt.positionInput=brca1&hgt.suggestTrack=knownGene&Submit=submit&hgsid=429339723_8sd4QD2jSAnAsa6cVCevtoOy4GAz&pix=1885

If instead of "genes" you do "transcripts", you will see 20 different

transcripts for this gene, including the one listed by NCBI.

I havent tried it yet (haven't upgraded R or bioconductor to latest
version), but there is now an Ensembl based annotation package as well,
that may work better??
http://bioconductor.org/packages/release/data/annotation/html/EnsDb.Hsapiens.v79.html

-Robert

On Wed, Jun 3, 2015 at 7:04 AM Ludwig Geistlinger <
Ludwig.Geistlinger at bio.ifi.lmu.de
<mailto:Ludwig.Geistlinger at bio.ifi.lmu.de>> wrote:

Dear Bioc annotation team,

Querying TxDb.Hsapiens.UCSC.hg38.knownGene for gene coordinates, e.g.
for

BRCA1; ENSG00000012048; entrez:672

via

genes(TxDb.Hsapiens.UCSC.hg38.knownGene, vals=list(gene_id="672"))

gives me:

GRanges object with 1 range and 1 metadata column: seqnames
ranges strand |     gene_id <Rle>            <IRanges>  <Rle> |
<character> 672    chr17 [43044295, 43170403]      - |         672
------- seqinfo: 455 sequences (1 circular) from hg38 genome


However, querying Ensembl and NCBI Gene
http://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000012048

http://www.ncbi.nlm.nih.gov/gene/672

the gene is located at (note the difference in the end position)

Chromosome 17: 43,044,295-43,125,483 reverse strand


How is the inconsistency explained and how to extract an ENSEMBL/NCBI
conform annotation from the TxDb object? (I am aware of biomaRt, but I
want to explicitely use the Bioc annotation functionality).

Thanks! Ludwig


-- Dipl.-Bioinf. Ludwig Geistlinger

Lehr- und Forschungseinheit f?r Bioinformatik Institut f?r Informatik
Ludwig-Maximilians-Universit?t M?nchen Amalienstrasse 17, 2. Stock, B?ro
A201 80333 M?nchen

Tel.: 089-2180-4067 eMail: Ludwig.Geistlinger at bio.ifi.lmu.de
<mailto:Ludwig.Geistlinger at bio.ifi.lmu.de>

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