Open lucaskbobadilla opened 4 years ago
Hi, Yes. It seems there is something weird in the annotation Can you extract exons from your TxDb object? You can check like this:
EX <- exons(TxDb) head(EX)
thanks a lot
Hi,
Here is the result for it:
`> EX <- exons(TxDb)
EX GRanges object with 168476 ranges and 1 metadata column: seqnames ranges strand | exon_id
| [1] asm.contigs_Scaffold_1 24476-24691 + | 1 [2] asm.contigs_Scaffold_1 25311-25377 + | 2 [3] asm.contigs_Scaffold_1 26086-26114 + | 3 [4] asm.contigs_Scaffold_1 26210-26418 + | 4 [5] asm.contigs_Scaffold_1 28111-28221 + | 5 ... ... ... ... . ... [168472] asm.contigs_Scaffold_9 33870715-33871005 - | 168472 [168473] asm.contigs_Scaffold_9 33873762-33873832 - | 168473 [168474] asm.contigs_Scaffold_9 33873975-33874063 - | 168474 [168475] asm.contigs_Scaffold_9 33874443-33874517 - | 168475 [168476] asm.contigs_Scaffold_9 33874599-33874942 - | 168476 ------- seqinfo: 16 sequences from an unspecified genome; no seqlengths`
Hi, Yes, it seems very complicated how genes and exons are annotated. do you think is any way to simplify? Specially for characteres like "-", "|", etc... For example, remove " asm.contigsScaffold" ,
thanks Estefi
I remove it still getting the same output.
I see. We should simplify in some way, because it is causing troubles for txDb objects. If you want, you can send me part of your gtf and I can try to explore a bit more your problem. thanks
Hi @estepi
I am facing the same issue.
* Number of extracted Genes = 42031
* Number of extracted Exon Bins = 158249
* Number of extracted intron bins = 129751
* Number of extracted trascripts = 42031
* Number of extracted junctions = 129751
* Number of AS bins (not include external) = 0
* Number of AS bins (include external) = 0
* Classified as:
ES bins = 0 (NaN%)
IR bins = 0 (NaN%)
Alt5'ss bins = 0 (NaN%)
Alt3'ss bins = 0 (NaN%)
Multiple AS bins = 0 (NaN%)
classified as:
ES bins = 0 (NaN%)
IR bins = 0 (NaN%)
Alt5'ss bins = 0 (NaN%)
Alt3'ss bins = 0 (NaN%)
EX <- exons(TxDb)
> EX
GRanges object with 171782 ranges and 1 metadata column:
seqnames ranges strand | exon_id
<Rle> <IRanges> <Rle> | <integer>
[1] CH398230.1 5010-5088 + | 1
[2] CH398230.1 6709-6764 + | 2
[3] CH398230.1 8466-8665 + | 3
[4] CH398230.1 8856-8898 + | 4
[5] CH398230.1 127667-128488 + | 5
... ... ... ... . ...
[171778] AAAA02050198.1 21-1187 + | 171778
[171779] AAAA02050204.1 733-1101 - | 171779
[171780] AAAA02050217.1 467-598 + | 171780
[171781] AAAA02050217.1 685-840 + | 171781
[171782] AAAA02050218.1 49-813 - | 171782
I obtained my gtf from here: ftp://ftp.ensemblgenomes.org/pub/plants/release-48/gtf/oryza_indica/
#!genome-build ASM465v1
#!genome-version ASM465v1
#!genome-date 2011-10
#!genome-build-accession GCA_000004655.2
#!genebuild-last-updated 2010-07
1 bgi gene 18113 20165 . + . gene_id "BGIOSGA002568"; gene_source "bgi"; gene_biotype "protein_coding";
1 bgi transcript 18113 20165 . + . gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding";
1 bgi exon 18113 19150 . + . gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; exon_number "1"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding"; exon_id "BGIOSGA002568-TA.1";
1 bgi CDS 18113 19150 . + 0 gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; exon_number "1"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding"; protein_id "BGIOSGA002568-PA";
1 bgi start_codon 18113 18115 . + 0 gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; exon_number "1"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding";
1 bgi exon 19344 20165 . + . gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; exon_number "2"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding"; exon_id "BGIOSGA002568-TA.2";
1 bgi CDS 19344 20162 . + 0 gene_id "BGIOSGA002568"; transcript_id "BGIOSGA002568-TA"; exon_number "2"; gene_source "bgi"; gene_biotype "protein_coding"; transcript_source "bgi"; transcript_biotype "protein_coding"; protein_id "BGIOSGA002568-PA";
Dear all,
Can you run this code and tell me what do you find?
library(GenomicFeatures) library(ASpli)
genomeTxDb <- makeTxDbFromGFF("Oryza_indica.ASM465v1.48.gtf")
features <- binGenome(genomeTxDb) head(features@bins) length(features@bins)
I checked Oryza annotation and I have the bins.
thanks a lot for your patience
Hi @estepi
I am getting genomic bins. However, I want to study Intron retention events and other AS events too. The output of your code is as follows
> genomeTxDb <- makeTxDbFromGFF("Oryza_indica.ASM465v1.48.gtf")
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Warning message:
In .get_cds_IDX(mcols0$type, mcols0$phase) :
The "phase" metadata column contains non-NA values for features of type
stop_codon. This information was ignored.
> features <- binGenome(genomeTxDb)
* Number of extracted Genes = 42031
* Number of extracted Exon Bins = 158249
* Number of extracted intron bins = 129751
* Number of extracted trascripts = 42031
* Number of extracted junctions = 129751
* Number of AS bins (not include external) = 0
* Number of AS bins (include external) = 0
* Classified as:
ES bins = 0 (NaN%)
IR bins = 0 (NaN%)
Alt5'ss bins = 0 (NaN%)
Alt3'ss bins = 0 (NaN%)
Multiple AS bins = 0 (NaN%)
classified as:
ES bins = 0 (NaN%)
IR bins = 0 (NaN%)
Alt5'ss bins = 0 (NaN%)
Alt3'ss bins = 0 (NaN%)
> head(features@bins)
GRanges object with 6 ranges and 8 metadata columns:
seqnames ranges strand | locus bin
<Rle> <IRanges> <Rle> | <character> <character>
BGIOSGA040744:E001 CH398230.1 5010-5088 + | BGIOSGA040744 E001
BGIOSGA040744:I001 CH398230.1 5089-6708 + | BGIOSGA040744 I001
BGIOSGA040744:E002 CH398230.1 6709-6764 + | BGIOSGA040744 E002
BGIOSGA040744:I002 CH398230.1 6765-8465 + | BGIOSGA040744 I002
BGIOSGA040744:E003 CH398230.1 8466-8665 + | BGIOSGA040744 E003
BGIOSGA040744:I003 CH398230.1 8666-8855 + | BGIOSGA040744 I003
feature symbol locus_overlap class
<character> <character> <character> <character>
BGIOSGA040744:E001 E BGIOSGA040744 - external
BGIOSGA040744:I001 I BGIOSGA040744 - -
BGIOSGA040744:E002 E BGIOSGA040744 - -
BGIOSGA040744:I002 I BGIOSGA040744 - -
BGIOSGA040744:E003 E BGIOSGA040744 - -
BGIOSGA040744:I003 I BGIOSGA040744 - -
event eventJ
<character> <character>
BGIOSGA040744:E001 external external
BGIOSGA040744:I001 - -
BGIOSGA040744:E002 - -
BGIOSGA040744:I002 - -
BGIOSGA040744:E003 - -
BGIOSGA040744:I003 - -
-------
seqinfo: 2140 sequences from an unspecified genome; no seqlengths
> length(features@bins)
[1] 288000
My concern is IR, ES and other bins show zero percentage. Is it normal to have these bins zero? I ran the examplary gtf and while binning I found these bins to be non-zero
> annFile <- aspliExampleGTF()
> aTxDb <- makeTxDbFromGFF(annFile)
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
> features <- binGenome( aTxDb )
* Number of extracted Genes = 10
* Number of extracted Exon Bins = 38
* Number of extracted intron bins = 34
* Number of extracted trascripts = 25
* Number of extracted junctions = 23
* Number of AS bins (not include external) = 17
* Number of AS bins (include external) = 17
* Classified as:
ES bins = 1 (6%)
IR bins = 3 (18%)
Alt5'ss bins = 2 (12%)
Alt3'ss bins = 2 (12%)
Multiple AS bins = 9 (53%)
classified as:
ES bins = 1 (11%)
IR bins = 4 (44%)
Alt5'ss bins = 2 (22%)
Alt3'ss bins = 1 (11%)
I am trying to understand if none of the bins were assigned as IR or ES or any other AS types, then will I get any IR or ES event being reported?
Hi @Rohit-Satyam, thanks for your reply
I see. No, it is not normal. If there is more than 1 isoform, we assume there are some bins that are supossed to be "alternative" This is our approach. I'm not familiar with your annotation, I should explore more and try to figure out why we are not finding AS bins.
What I would try is to continue with the pipeline and see if you can detect intron retention, even though introns are not labelled as IR. Can you try this in the meantime?
Thanks
Sure. I will give it a shot. Thanks for your prompt response. I really appreciate your time.
Hi @estepi
I had this feeling that such behavior could only happen if for every gene there is exactly a single transcript in the gtf file. I went back to check if that's the issue. And I think in Osativa indica gtf, that is the actual issue. I also tried running the same steps with the Osativa japonica gtf and it runs fine. Can you confirm, if it's the gtf that deserves all the blame
> library(ASpli)
> library(GenomicFeatures)
> genomeTxDb <- makeTxDbFromGFF("Oryza_sativa.IRGSP-1.0.48.gtf")
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Warning message:
In .get_cds_IDX(mcols0$type, mcols0$phase) :
The "phase" metadata column contains non-NA values for features of type stop_codon. This
information was ignored.
> features <- binGenome(genomeTxDb)
* Number of extracted Genes = 38978
* Number of extracted Exon Bins = 167945
* Number of extracted intron bins = 128442
* Number of extracted trascripts = 45772
* Number of extracted junctions = 126775
* Number of AS bins (not include external) = 3424
* Number of AS bins (include external) = 3463
* Classified as:
ES bins = 283 (8%)
IR bins = 2039 (60%)
Alt5'ss bins = 345 (10%)
Alt3'ss bins = 592 (17%)
Multiple AS bins = 165 (5%)
classified as:
ES bins = 29 (18%)
IR bins = 57 (35%)
Alt5'ss bins = 31 (19%)
Alt3'ss bins = 40 (24%)
Hi @Rohit-Satyam, great news ! :)
It can happen because annotation is very young, isnt it?
Anyway, you can still analyze IR despite this miss-annotation and see what happens...And maybe you will be the firts to systematically describe AS events in this specie ;)
thanks for your post
Hello,
I am having some weird results after running binGenome. I am following the example analysis.
First I build the txDb as described using genomicFeature package:
# build a TxDb of the genome (uses genomeFeatures package)
TxDb <- makeTxDbFromGFF( file="WH_augustus_chr_only.genes.gff3", format="gff3")
Import genomic features from the file as a GRanges object ... OK Prepare the 'metadata' data frame ... OK Make the TxDb object ... OK
No problems with that. But when I try to generate the features using:
features <- binGenome(TxDb) # extract features from annotation
I am getting this output:
Why I am not getting any AS bins? Is there something missing in the gff file? or is there any step that I missed in the txDb step?
Thank you!