The GenomicFeatures package implements the TxDb container for
storing transcript metadata for a given organism. A TxDb object
stores the genomic positions of the 5’ and 3’ untranslated regions
(UTRs), protein coding sequences (CDSs), and exons for a set of mRNA
transcripts. The genomic positions are stored and reported with respect
to a given genome assembly. TxDb objects have numerous accessors
functions to allow such features to be retrieved individually or grouped
together in a way that reflects the underlying biology.
All TxDb objects are backed by a SQLite database that stores
the genomic positions and relationships between pre-processed mRNA
transcripts, exons, protein coding sequences, and their related
gene identifiers.
GenomicFeatures packageInstall the package with:
if (!require("BiocManager", quietly=TRUE))
install.packages("BiocManager")
BiocManager::install("GenomicFeatures")
Then load it with:
suppressPackageStartupMessages(library(GenomicFeatures))
## Error in library(GenomicFeatures): there is no package called 'GenomicFeatures'
TxDb objectThere are three ways that users can obtain a TxDb object.
One way is to use the loadDb method to load the object directly
from an appropriate .sqlite database file.
Here we are loading a previously created TxDb object
based on UCSC known gene data. This database only contains a small
subset of the possible annotations for human and is only included to
demonstrate and test the functionality of the
GenomicFeatures package as a demonstration.
samplefile <- system.file("extdata", "hg19_knownGene_sample.sqlite",
package="GenomicFeatures")
txdb <- loadDb(samplefile)
## Error in loadDb(samplefile): could not find function "loadDb"
txdb
## Error: object 'txdb' not found
In this case, the TxDb object has been returned by
the loadDb method.
More commonly however, we expect that users will just load a TxDb annotation package like this:
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
## Error in library(TxDb.Hsapiens.UCSC.hg19.knownGene): there is no package called 'TxDb.Hsapiens.UCSC.hg19.knownGene'
hg19_txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene # shorthand (for convenience)
## Error: object 'TxDb.Hsapiens.UCSC.hg19.knownGene' not found
hg19_txdb
## Error: object 'hg19_txdb' not found
Loading the package like this will also create a TxDb
object, and by default that object will have the same name as the
package itself.
Finally, the third way to obtain a TxDb object is to use one of
the numerous tools defined in the txdbmaker package. txdbmaker
provides a set of tools for making TxDb objects from genomic
annotations from various sources (e.g. UCSC, Ensembl, and GFF files).
See the vignette in the txdbmaker package for more information.
TxDb objectIt is possible to filter the data that is returned from a
TxDb object based on it’s chromosome. This can be a
useful way to limit the things that are returned if you are only
interested in studying a handful of chromosomes.
To determine which chromosomes are currently active, use the
seqlevels method. For example:
head(seqlevels(hg19_txdb))
## Error in seqlevels(hg19_txdb): could not find function "seqlevels"
Will tell you all the chromosomes that are active for the
TxDb.Hsapiens.UCSC.hg19.knownGene TxDb object (by
default it will be all of them).
If you then wanted to only set Chromosome 1 to be active you could do it like this:
seqlevels(hg19_txdb) <- "chr1"
## Error: object 'hg19_txdb' not found
So if you ran this, then from this point on in your R session only
chromosome 1 would be consulted when you call the various retrieval
methods… If you need to reset back to the original seqlevels (i.e.
to the seqlevels stored in the db), then set the seqlevels to
seqlevels0(hg19_txdb).
seqlevels(hg19_txdb) <- seqlevels0(hg19_txdb)
## Error in seqlevels0(hg19_txdb): could not find function "seqlevels0"
Exercise:
Use seqlevels to set only chromsome 15 to be active. BTW,
the rest of this vignette will assume you have succeeded at this.
Solution:
seqlevels(hg19_txdb) <- "chr15"
## Error: object 'hg19_txdb' not found
seqlevels(hg19_txdb)
## Error in seqlevels(hg19_txdb): could not find function "seqlevels"
select() methodThe TxDb objects inherit from AnnotationDb
objects (just as the ChipDb and OrgDb objects do).
One of the implications of this relationship is that these object
ought to be used in similar ways to each other. Therefore we have
written supporting columns, keytypes, keys
and select methods for TxDb objects.
These methods can be a useful way of extracting data from a
TxDb object. And they are used in the same way that
they would be used to extract information about a ChipDb or
an OrgDb object. Here is a simple example of how to find the
UCSC transcript names that match with a set of gene IDs.
keys <- c("100033416", "100033417", "100033420")
columns(hg19_txdb)
## Error in columns(hg19_txdb): could not find function "columns"
keytypes(hg19_txdb)
## Error in keytypes(hg19_txdb): could not find function "keytypes"
select(hg19_txdb, keys = keys, columns="TXNAME", keytype="GENEID")
## Error in select(hg19_txdb, keys = keys, columns = "TXNAME", keytype = "GENEID"): could not find function "select"
Exercise: For the genes in the example above, find the chromosome and strand information that will go with each of the transcript names.
Solution:
columns(hg19_txdb)
## Error in columns(hg19_txdb): could not find function "columns"
cols <- c("TXNAME", "TXSTRAND", "TXCHROM")
select(hg19_txdb, keys=keys, columns=cols, keytype="GENEID")
## Error in select(hg19_txdb, keys = keys, columns = cols, keytype = "GENEID"): could not find function "select"
GRanges objectsRetrieving data with select is useful, but sometimes it is more
convenient to extract the result as a GRanges object. This is
often the case when you are doing counting or specialized overlap
operations downstream. For these use cases there is another family of
methods available.
Perhaps the most common operations for a TxDb object
is to retrieve the genomic coordinates or ranges for exons,
transcripts or coding sequences. The functions
transcripts, exons, and cds return
the coordinate information as a GRanges object.
As an example, all transcripts present in a TxDb object
can be obtained as follows:
GR <- transcripts(hg19_txdb)
## Error in transcripts(hg19_txdb): could not find function "transcripts"
GR[1:3]
## Error: object 'GR' not found
The transcripts function returns a GRanges class
object. You can learn a lot more about the manipulation of these
objects by reading the GenomicRanges introductory
vignette. The show method for a GRanges object
will display the ranges, seqnames (a chromosome or a contig), and
strand on the left side and then present related metadata on the right
side.
The strand function is used to obtain the strand
information from the transcripts. The sum of the Lengths of the
Rle object that strand returns is equal to the
length of the GRanges object.
tx_strand <- strand(GR)
## Error in strand(GR): could not find function "strand"
tx_strand
## Error: object 'tx_strand' not found
sum(runLength(tx_strand))
## Error in runLength(tx_strand): could not find function "runLength"
length(GR)
## Error: object 'GR' not found
In addition, the transcripts function can also be used to
retrieve a subset of the transcripts available such as those on the
+-strand of chromosome 1.
GR <- transcripts(hg19_txdb, filter=list(tx_chrom = "chr15", tx_strand = "+"))
## Error in transcripts(hg19_txdb, filter = list(tx_chrom = "chr15", tx_strand = "+")): could not find function "transcripts"
length(GR)
## Error: object 'GR' not found
unique(strand(GR))
## Error in strand(GR): could not find function "strand"
The exons and cds functions can also be used
in a similar fashion to retrive genomic coordinates for exons and
coding sequences.
The promoters function computes a GRanges object
that spans the promoter region around the transcription start site
for the transcripts in a TxDb object. The upstream
and downstream arguments define the number of bases upstream
and downstream from the transcription start site that make up the
promoter region.
PR <- promoters(hg19_txdb, upstream=2000, downstream=400)
## Error in promoters(hg19_txdb, upstream = 2000, downstream = 400): could not find function "promoters"
PR
## Error: object 'PR' not found
A similar function (terminators) is provided to compute the
terminator region around the transcription end site for the
transcripts in a TxDb object.
Exercise:
Use exons to retrieve all the exons from chromosome 15.
How does the length of this compare to the value returned by
transcripts?
Solution:
EX <- exons(hg19_txdb)
## Error in exons(hg19_txdb): could not find function "exons"
EX[1:4]
## Error: object 'EX' not found
length(EX)
## Error: object 'EX' not found
length(GR)
## Error: object 'GR' not found
Often one is interested in how particular genomic features relate to
each other, and not just their genomic positions. For example, it might
be of interest to group transcripts by gene or to group exons by transcript.
Such groupings are supported by the transcriptsBy,
exonsBy, and cdsBy functions.
The following call can be used to group transcripts by genes:
GRList <- transcriptsBy(hg19_txdb, by = "gene")
## Error in transcriptsBy(hg19_txdb, by = "gene"): could not find function "transcriptsBy"
length(GRList)
## Error: object 'GRList' not found
names(GRList)[10:13]
## Error: object 'GRList' not found
GRList[11:12]
## Error: object 'GRList' not found
The transcriptsBy function returns a GRangesList
class object. As with GRanges objects, you can learn more
about these objects by reading the GenomicRanges
introductory vignette. The show method for a
GRangesList object will display as a list of GRanges
objects. And, at the bottom the seqinfo will be displayed once for
the entire list.
For each of these three functions, there is a limited set of options
that can be passed into the by argument to allow grouping.
For the transcriptsBy function, you can group by gene,
exon or cds, whereas for the exonsBy and cdsBy
functions can only be grouped by transcript (tx) or gene.
So as a further example, to extract all the exons for each transcript you can call:
GRList <- exonsBy(hg19_txdb, by = "tx")
## Error in exonsBy(hg19_txdb, by = "tx"): could not find function "exonsBy"
length(GRList)
## Error: object 'GRList' not found
names(GRList)[10:13]
## Error: object 'GRList' not found
GRList[[12]]
## Error: object 'GRList' not found
As you can see, the GRangesList objects returned from each
function contain genomic positions and identifiers grouped into a
list-like object according to the type of feature specified in the by
argument. The object returned can then be used by functions like
findOverlaps to contextualize alignments from
high-throughput sequencing.
The identifiers used to label the GRanges objects depend upon
the data source used to create the TxDb object. So
the list identifiers will not always be Entrez Gene IDs, as they were
in the first example. Furthermore, some data sources do not provide a
unique identifier for all features. In this situation, the group
label will be a synthetic ID created by GenomicFeatures to
keep the relations between features consistent in the database this
was the case in the 2nd example. Even though the results will
sometimes have to come back to you as synthetic IDs, you can still
always retrieve the original IDs.
Exercise:
Starting with the tx_ids that are the names of the GRList object we
just made, use select to retrieve that matching transcript
names. Remember that the list used a by argument = “tx”, so
the list is grouped by transcript IDs.
Solution:
GRList <- exonsBy(hg19_txdb, by = "tx")
## Error in exonsBy(hg19_txdb, by = "tx"): could not find function "exonsBy"
tx_ids <- names(GRList)
## Error: object 'GRList' not found
head(select(hg19_txdb, keys=tx_ids, columns="TXNAME", keytype="TXID"))
## Error in select(hg19_txdb, keys = tx_ids, columns = "TXNAME", keytype = "TXID"): could not find function "select"
Finally, the order of the results in a GRangesList object can
vary with the way in which things were grouped. In most cases the
grouped elements of the GRangesList object will be listed in
the order that they occurred along the chromosome. However, when
exons or CDS parts are grouped by transcript, they will instead be
grouped according to their position along the transcript itself.
This is important because alternative splicing can mean that the
order along the transcript can be different from that along the
chromosome.
The intronsByTranscript, fiveUTRsByTranscript
and threeUTRsByTranscript are convenience functions that
provide behavior equivalent to the grouping functions, but in
prespecified form. These functions return a GRangesList
object grouped by transcript for introns, 5’ UTR’s, and 3’ UTR’s,
respectively. Below are examples of how you can call these methods.
length(intronsByTranscript(hg19_txdb))
## Error in intronsByTranscript(hg19_txdb): could not find function "intronsByTranscript"
length(fiveUTRsByTranscript(hg19_txdb))
## Error in fiveUTRsByTranscript(hg19_txdb): could not find function "fiveUTRsByTranscript"
length(threeUTRsByTranscript(hg19_txdb))
## Error in threeUTRsByTranscript(hg19_txdb): could not find function "threeUTRsByTranscript"
The GenomicFeatures package also provides functions for
converting from ranges to actual sequence (when paired
with an appropriate BSgenome package).
suppressPackageStartupMessages(library(BSgenome.Hsapiens.UCSC.hg19))
## Error in library(BSgenome.Hsapiens.UCSC.hg19): there is no package called 'BSgenome.Hsapiens.UCSC.hg19'
genome <- BSgenome.Hsapiens.UCSC.hg19 # shorthand (for convenience)
## Error: object 'BSgenome.Hsapiens.UCSC.hg19' not found
tx_seqs1 <- extractTranscriptSeqs(genome, hg19_txdb, use.names=TRUE)
## Error in extractTranscriptSeqs(genome, hg19_txdb, use.names = TRUE): could not find function "extractTranscriptSeqs"
And, once these sequences have been extracted, you can translate them
into proteins with translate:
suppressWarnings(translate(tx_seqs1))
## Error in translate(tx_seqs1): could not find function "translate"
Exercise:
But of course this is not a meaningful translation, because the call
to extractTranscriptSeqs will have extracted all
the transcribed regions of the genome regardless of whether or not
they are translated. Look at the manual page for
extractTranscriptSeqs and see how you can use cdsBy
to only translate only the coding regions.
Solution:
cds_seqs <- extractTranscriptSeqs(Hsapiens,
cdsBy(hg19_txdb, by="tx", use.names=TRUE))
## Error in extractTranscriptSeqs(Hsapiens, cdsBy(hg19_txdb, by = "tx", use.names = TRUE)): could not find function "extractTranscriptSeqs"
translate(cds_seqs)
## Error in translate(cds_seqs): could not find function "translate"
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