| Title: | Transcript Quantification Import with Automatic Metadata |
|---|---|
| Description: | Transcript quantification import from Salmon and other quantifiers with automatic attachment of transcript ranges and release information, and other associated metadata. De novo transcriptomes can be linked to the appropriate sources with linkedTxomes and shared for computational reproducibility. |
| Authors: | Michael Love [aut, cre], Charlotte Soneson [aut, ctb], Peter Hickey [aut, ctb], Rob Patro [aut, ctb], NIH NHGRI [fnd], CZI [fnd] |
| Maintainer: | Michael Love <[email protected]> |
| License: | GPL-2 |
| Version: | 1.25.0 |
| Built: | 2024-10-31 06:30:31 UTC |
| Source: | https://github.com/bioc/tximeta |
The tximeta package imports abundances (TPM), estimated counts,
and effective lengths from Salmon, alevin, piscem or other quantification
tools, and will output a SummarizedExperiment object. For
Salmon / alevin / piscem quantification data, tximeta will
try to identify the correct provenance of the reference transcripts
and automatically attach the transcript ranges to the
SummarizedExperiment, to facilitate downstream integration with
other datasets. The automatic identification of reference transcripts
should work out-of-the-box for human or mouse transcriptomes from
the sources: GENCODE, Ensembl, or RefSeq.
The main functions are:
tximeta - with key argument: coldata
summarizeToGene,SummarizedExperiment-method - summarize quants to gene-level
retrieveDb - retrieve the transcript database
addIds - add transcript or gene ID (see gene argument)
All software-related questions should be posted to the Bioconductor Support Site:
https://support.bioconductor.org
The code can be viewed at the GitHub repository, which also lists the contributor code of conduct:
https://github.com/thelovelab/tximeta
Michael I. Love, Charlotte Soneson, Peter Hickey, Rob Patro
tximeta reference:
Michael I. Love, Charlotte Soneson, Peter F. Hickey, Lisa K. Johnson N. Tessa Pierce, Lori Shepherd, Martin Morgan, Rob Patro (2020) Tximeta: reference sequence checksums for provenance identification in RNA-seq. PLOS Computational Biology. https://doi.org/10.1371/journal.pcbi.1007664
tximport reference (the effective length offset and counts-from-abundance):
Charlotte Soneson, Michael I. Love, Mark D. Robinson (2015) Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. http://doi.org/10.12688/f1000research.7563
Useful links:
Working similarly to addExons, this function
can be used to add information about CDS (coding sequence)
to the SummarizedExperiment object. As not all transcripts
are coding, we have CDS information for only a subset of the
rows of the object. For this reason, a logical indicator for
whether the transcript is coding, mcols(se)$coding,
is added as a column to the metadata columns of the rowRanges
of the object. An additional column, mcols(se)$cds,
is added to the metadata columns, which is a GRangesList
with either the CDS regions (if the transcript is coding),
or the original transcript/exon ranges (if the transcript is non-coding).
This is necessary, as GRangesList cannot have NA elements.
As with addExons, this function is designed only
for transcript-level objects.
addCDS(se)addCDS(se)
se |
the SummarizedExperiment |
a SummarizedExperiment
After running tximeta, the SummarizedExperiment output
will have GRanges representing the transcript locations
attached as rowRanges to the object. These provide the
start and end of the transcript in the genomic coordiantes, and
strand information. However, the exonic locations are not provided.
This function, addExons, swaps out the GRanges
with a GRangesList, essentially a list along the rows of the
SummarizedExperiment, where each element of the list is a
GRanges providing the locations of the exons for that transcript.
addExons(se)addExons(se)
se |
the SummarizedExperiment |
This function is designed only for transcript-level objects.
This "lack of a feature" reflects a belief on the part of the package author
that it makes more sense to think about exons belonging to transcripts
than to genes. For users desiring exonic information alongside
gene-level objects, for example, which exons are associated with
a particular gene, it is recommended to pull out the relevant
GRangesList for the transcripts of this gene, while the object
represents transcript-level data, such that the exons are still
associated with transcripts.
For an example of addExons, please see the tximeta vignette.
a SummarizedExperiment
For now this function just works with SummarizedExperiments
with Ensembl gene or transcript IDs. See example
of usage in tximeta vignette. For obtaining
multiple matching IDs for each row of the SummarizedExperiment
set multiVals="list". See select for documentation
on use of multiVals.
addIds(se, column, fromDb = FALSE, gene = FALSE, ...)addIds(se, column, fromDb = FALSE, gene = FALSE, ...)
se |
the SummarizedExperiment |
column |
the name of the new ID to add (a |
fromDb |
logical, whether to use the TxDb/EnsDb that is associated
with |
gene |
logical, whether to map by genes or transcripts (default is FALSE).
if rows are genes, and easily detected as such (ENSG or ENSMUSG), it will
automatically switch to TRUE. if rows are transcripts and |
... |
arguments passed to |
a SummarizedExperiment
example(tximeta) library(org.Dm.eg.db) se <- addIds(se, "REFSEQ", gene=FALSE)example(tximeta) library(org.Dm.eg.db) se <- addIds(se, "REFSEQ", gene=FALSE)
Running getTximetaBFC will report the saved directory,
if it has been determined, or will return NULL.
Running setTximetaBFC will ask the user to specify a
BiocFileCache directory for accessing and saving TxDb sqlite files.
Note that tximeta's BiocFileCache can be set by the environmental
variable TXIMETA_HUB_CACHE, which will reset the cache location.
getTximetaBFC() setTximetaBFC(dir, quiet = FALSE)getTximetaBFC() setTximetaBFC(dir, quiet = FALSE)
dir |
the location for tximeta's BiocFileCache. can be missing
in which case the function will call |
quiet |
whether to suppress feedback message |
the directory of the BiocFileCache used by tximeta
(or nothing, in the case of setTximetaBFC)
# getting the BiocFileCache used by tximeta # (may not be set, which uses BiocFileCache default or temp directory) getTximetaBFC() # don't want to actually change user settings so this is not run: # setTximetaBFC()# getting the BiocFileCache used by tximeta # (may not be set, which uses BiocFileCache default or temp directory) getTximetaBFC() # don't want to actually change user settings so this is not run: # setTximetaBFC()
makeLinkedTxome reads the digest associated with a Salmon
index at indexDir, and links it to key information
about the transcriptome, including the source, organism,
release, and genome (these are custom character strings),
as well as the locations (e.g. local, HTTP, or FTP) for one or more fasta
files and one gtf file. loadLinkedTxome loads this
information from a JSON file. See Details.
makeLinkedTxome( indexDir, source, organism, release, genome, fasta, gtf, write = TRUE, jsonFile ) loadLinkedTxome(jsonFile)makeLinkedTxome( indexDir, source, organism, release, genome, fasta, gtf, write = TRUE, jsonFile ) loadLinkedTxome(jsonFile)
indexDir |
the local path to the Salmon index |
source |
the source of transcriptome (e.g. "de-novo"). Note: if you specify "GENCODE" or "Ensembl", this will trigger behavior by tximeta that may not be desired: e.g. attempts to download canonical transcriptome data from AnnotationHub (unless useHub=FALSE when running tximeta) and parsing of Ensembl GTF using ensembldb (which may fail if the GTF file has been modified). For transcriptomes that are defined by local GTF files, it is recommended to use the terms "LocalGENCODE" or "LocalEnsembl". Setting "LocalEnsembl" will also strip version numbers from the FASTA transcript IDs to enable matching with the Ensembl GTF. |
organism |
organism (e.g. "Homo sapiens") |
release |
release number (e.g. "27") |
genome |
genome (e.g. "GRCh38", or "none") |
fasta |
location(s) for the FASTA transcript sequences (of which the transcripts used to build the index is equal or a subset). This can be a local path, or an HTTP or FTP URL |
gtf |
location for the GTF/GFF file
(of which the transcripts used to build the index is equal or a subset).
This can be a local path, or an HTTP or FTP URL
While the |
write |
logical, should a JSON file be written out which documents the transcriptome digest and metadata? (default is TRUE) |
jsonFile |
the path to the json file for the linkedTxome |
makeLinkedTxome links the information about the transcriptome
used for quantification in two ways:
1) the function will store a record in tximeta's cache such that
future import of quantification data will automatically access and
parse the GTF as if the transcriptome were one of those automatically
detected by tximeta. Then all features of tximeta (e.g. summarization
to gene, programmatic adding of IDs or metadata) will be available;
2) it will by default write out a JSON file
that can be shared, or posted online, and which can be read by
loadLinkedTxome which will store the information in tximeta's
cache. This should make the full quantification-import pipeline
computationally reproducible / auditable even for transcriptomes
which differ from those provided by references (GENCODE, Ensembl,
RefSeq).
For further details please see the "Linked transcriptomes" section of the tximeta vignette.
nothing, the function is run for its side effects
# point to a Salmon quantification file with an additional artificial transcript dir <- system.file("extdata/salmon_dm", package="tximportData") file <- file.path(dir, "SRR1197474.plus", "quant.sf") coldata <- data.frame(files=file, names="SRR1197474", sample="1", stringsAsFactors=FALSE) # now point to the Salmon index itself to create a linkedTxome # as the index will not match a known txome indexDir <- file.path(dir, "Dm.BDGP6.22.98.plus_salmon-0.14.1") # point to the source FASTA and GTF: fastaFTP <- c("ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/cdna/Drosophila_melanogaster.BDGP6.22.cdna.all.fa.gz", "ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/ncrna/Drosophila_melanogaster.BDGP6.22.ncrna.fa.gz", "extra_transcript.fa.gz") gtfPath <- file.path(dir, "Drosophila_melanogaster.BDGP6.22.98.plus.gtf.gz") # now create a linkedTxome, linking the Salmon index to its FASTA and GTF sources makeLinkedTxome(indexDir=indexDir, source="Ensembl", organism="Drosophila melanogaster", release="98", genome="BDGP6.22", fasta=fastaFTP, gtf=gtfPath, write=FALSE) # to clear the entire linkedTxome table # (don't run unless you want to clear this table!) # bfcloc <- getTximetaBFC() # bfc <- BiocFileCache(bfcloc) # bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)# point to a Salmon quantification file with an additional artificial transcript dir <- system.file("extdata/salmon_dm", package="tximportData") file <- file.path(dir, "SRR1197474.plus", "quant.sf") coldata <- data.frame(files=file, names="SRR1197474", sample="1", stringsAsFactors=FALSE) # now point to the Salmon index itself to create a linkedTxome # as the index will not match a known txome indexDir <- file.path(dir, "Dm.BDGP6.22.98.plus_salmon-0.14.1") # point to the source FASTA and GTF: fastaFTP <- c("ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/cdna/Drosophila_melanogaster.BDGP6.22.cdna.all.fa.gz", "ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/ncrna/Drosophila_melanogaster.BDGP6.22.ncrna.fa.gz", "extra_transcript.fa.gz") gtfPath <- file.path(dir, "Drosophila_melanogaster.BDGP6.22.98.plus.gtf.gz") # now create a linkedTxome, linking the Salmon index to its FASTA and GTF sources makeLinkedTxome(indexDir=indexDir, source="Ensembl", organism="Drosophila melanogaster", release="98", genome="BDGP6.22", fasta=fastaFTP, gtf=gtfPath, write=FALSE) # to clear the entire linkedTxome table # (don't run unless you want to clear this table!) # bfcloc <- getTximetaBFC() # bfc <- BiocFileCache(bfcloc) # bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)
A simple wrapper function for constructing a DGEList for use with edgeR. See vignette for an example. Requires installation of the edgeR package from Bioconductor.
makeDGEList(se)makeDGEList(se)
se |
a SummarizedExperiment produced by tximeta |
a DGEList
This helper function retrieves the cDNA sequence of the transcripts used for expression quantification. This function either downloads or loads the transcript sequence from cache, it does not re-order or check against the rows of the SummarizedExperiment (which could be already summarized to genes for example).
retrieveCDNA(se, quiet = FALSE)retrieveCDNA(se, quiet = FALSE)
se |
the SummarizedExperiment |
quiet |
logical, suppress messages |
a DNAStringSet object
## Not run: # this example is not run because it requires access to Ensembl ftp example(tximeta) cdna <- retrieveCDNA(se) ## End(Not run)## Not run: # this example is not run because it requires access to Ensembl ftp example(tximeta) cdna <- retrieveCDNA(se) ## End(Not run)
SummarizedExperiment objects returned by tximeta have
associated TxDb or EnsDb databases which are cached locally and used
to perform various metadata related tasks. This helper function
retrieves the database itself for the user to perform any additional
operations.
retrieveDb(se)retrieveDb(se)
se |
the SummarizedExperiment |
a database object
example(tximeta) edb <- retrieveDb(se)example(tximeta) edb <- retrieveDb(se)
Construct a new SummarizedExperiment by splitting one of the assays
into a list of assays, each of which contains features of a given
'type'. It is assumed that there is a one-to-one correspondence
between feature sets of different types; for example, these can be
spliced and unspliced variants of the same transcripts. The type of
each feature in the original SummarizedExperiment, and the correspondence
between the features of different types, are given in a data.frame.
splitSE(se, splitDf, assayName)splitSE(se, splitDf, assayName)
se |
A |
splitDf |
A |
assayName |
A character scalar, indicating the assay of |
A SummarizedExperiment object with the same columns
as the input object, and the same number of assays as the number
of columns in splitDf. The assays will be named by the
column names of splitDf. The colData and metadata
of the input SummarizedExperiment object are copied to the
output object. The row names are set to the feature IDs in the
first column of splitDf.
se <- SummarizedExperiment::SummarizedExperiment( assays = S4Vectors::SimpleList( counts = as(matrix(1:15, nrow = 5), "sparseMatrix"), logcounts = log2(matrix(1:15, nrow = 5)) ), colData = S4Vectors::DataFrame(sID = paste0("S", 1:3), condition = c("A", "A", "B")), metadata = list(md1 = "annotation") ) rownames(se) <- paste0("G", 1:5) colnames(se) <- paste0("P", 1:3) splitDf <- data.frame(spliced = c("G1", "G2", "G6"), unspliced = c("G3", "G5", "G4"), stringsAsFactors = FALSE) splse <- splitSE(se = se, splitDf = splitDf, assayName = "counts")se <- SummarizedExperiment::SummarizedExperiment( assays = S4Vectors::SimpleList( counts = as(matrix(1:15, nrow = 5), "sparseMatrix"), logcounts = log2(matrix(1:15, nrow = 5)) ), colData = S4Vectors::DataFrame(sID = paste0("S", 1:3), condition = c("A", "A", "B")), metadata = list(md1 = "annotation") ) rownames(se) <- paste0("G", 1:5) colnames(se) <- paste0("P", 1:3) splitDf <- data.frame(spliced = c("G1", "G2", "G6"), unspliced = c("G3", "G5", "G4"), stringsAsFactors = FALSE) splse <- splitSE(se = se, splitDf = splitDf, assayName = "counts")
Summarizes abundances, counts, lengths, (and inferential
replicates or variance) from transcript- to gene-level.
Transcript IDs are stored as a CharacterList in the mcols
of the output object.
This function operates on SummarizedExperiment objects, and
will automatically access the relevant TxDb (by either finding it
in the BiocFileCache or by building it from an ftp location).
This function uses the tximport package to perform summarization,
where a method is defined that works on simple lists.
## S4 method for signature 'SummarizedExperiment' summarizeToGene( object, assignRanges = c("range", "abundant"), varReduce = FALSE, ... )## S4 method for signature 'SummarizedExperiment' summarizeToGene( object, assignRanges = c("range", "abundant"), varReduce = FALSE, ... )
object |
a SummarizedExperiment produced by |
assignRanges |
|
varReduce |
whether to reduce per-sample inferential replicates
information into a matrix of sample variances |
... |
arguments passed to |
a SummarizedExperiment with summarized quantifications
and transcript IDs as a CharacterList in the mcols
example(tximeta) gse <- summarizeToGene(se)example(tximeta) gse <- summarizeToGene(se)
tximeta leverages the hashed digest of the Salmon or piscem index,
in addition to a number of core Bioconductor packages (GenomicFeatures,
ensembldb, AnnotationHub, GenomeInfoDb, BiocFileCache) to automatically
populate metadata for the user, without additional effort from the user.
For other quantifiers see the customMetaInfo argument below.
tximeta( coldata, type = NULL, txOut = TRUE, skipMeta = FALSE, skipSeqinfo = FALSE, useHub = TRUE, markDuplicateTxps = FALSE, cleanDuplicateTxps = FALSE, customMetaInfo = NULL, skipFtp = FALSE, ... )tximeta( coldata, type = NULL, txOut = TRUE, skipMeta = FALSE, skipSeqinfo = FALSE, useHub = TRUE, markDuplicateTxps = FALSE, cleanDuplicateTxps = FALSE, customMetaInfo = NULL, skipFtp = FALSE, ... )
coldata |
a data.frame with at least two columns (others will propogate to object):
if |
type |
what quantifier was used (see |
txOut |
whether to output transcript-level data.
|
skipMeta |
whether to skip metadata generation
(e.g. to avoid errors if not connected to internet).
This calls |
skipSeqinfo |
whether to skip the addition of Seqinfo, which requires an internet connection to download the relevant chromosome information table from UCSC |
useHub |
whether to first attempt to download a TxDb/EnsDb
object from AnnotationHub, rather than creating from a
GTF file from FTP (default is TRUE). If FALSE, it will
force |
markDuplicateTxps |
whether to mark the status
( |
cleanDuplicateTxps |
whether to try to clean duplicate transcripts (exact sequence duplicates) by replacing the transcript names that do not appear in the GTF with those that do appear in the GTF |
customMetaInfo |
the relative path to a custom metadata
information JSON file, relative to the paths in |
skipFtp |
whether to avoid |
... |
arguments passed to |
Most of the code in tximeta works to add metadata and transcript ranges
when the quantification was performed with Salmon. However,
tximeta can be used with any quantification type that is supported
by tximport, where it will return an non-ranged SummarizedExperiment.
tximeta performs a lookup of the hashed digest of the index
(stored in an auxilary information directory of the Salmon output)
against a database of known transcriptomes, which lives within the tximeta
package and is continually updated on Bioconductor's release schedule.
In addition, tximeta performs a lookup of the digest against a
locally stored table of linkedTxome's (see link{makeLinkedTxome}).
If tximeta detects a match, it will automatically populate,
e.g. the transcript locations, the transcriptome release,
the genome with correct chromosome lengths, etc. It allows for automatic
and correct summarization of transcript-level quantifications to the gene-level
via summarizeToGene without the need to manually build
a tx2gene table.
tximeta on the first run will ask where the BiocFileCache for
this package should be kept, either using a default location or a temporary
directory. At any point, the user can specify a location using
setTximetaBFC and this choice will be saved for future sessions.
Multiple users can point to the same BiocFileCache, such that
transcript databases (TxDb or EnsDb) associated with certain Salmon indices
and linkedTxomes can be accessed by different users without additional
effort or time spent downloading and building the relevant TxDb / EnsDb.
Note that, if the TxDb or EnsDb is present in AnnotationHub, tximeta will
use this object instead of downloading and building a TxDb/EnsDb from GTF
(to disable this set useHub=FALSE).
In order to allow that multiple users can read and write to the same location, one should set the BiocFileCache directory to have group write permissions (g+w).
a SummarizedExperiment with metadata on the rowRanges.
(if the hashed digest in the Salmon or Sailfish index does not match
any known transcriptomes, or any locally saved linkedTxome,
tximeta will just return a non-ranged SummarizedExperiment)
# point to a Salmon quantification file: dir <- system.file("extdata/salmon_dm", package="tximportData") files <- file.path(dir, "SRR1197474", "quant.sf") coldata <- data.frame(files, names="SRR1197474", condition="A", stringsAsFactors=FALSE) # normally we would just run the following which would download the appropriate metadata # se <- tximeta(coldata) # for this example, we instead point to a local path where the GTF can be found # by making a linkedTxome: indexDir <- file.path(dir, "Dm.BDGP6.22.98_salmon-0.14.1") fastaFTP <- c("ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/cdna/Drosophila_melanogaster.BDGP6.22.cdna.all.fa.gz", "ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/ncrna/Drosophila_melanogaster.BDGP6.22.ncrna.fa.gz") gtfPath <- file.path(dir, "Drosophila_melanogaster.BDGP6.22.98.gtf.gz") makeLinkedTxome(indexDir=indexDir, source="LocalEnsembl", organism="Drosophila melanogaster", release="98", genome="BDGP6.22", fasta=fastaFTP, gtf=gtfPath, write=FALSE) se <- tximeta(coldata) # to clear the entire linkedTxome table # (don't run unless you want to clear this table!) # bfcloc <- getTximetaBFC() # bfc <- BiocFileCache(bfcloc) # bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)# point to a Salmon quantification file: dir <- system.file("extdata/salmon_dm", package="tximportData") files <- file.path(dir, "SRR1197474", "quant.sf") coldata <- data.frame(files, names="SRR1197474", condition="A", stringsAsFactors=FALSE) # normally we would just run the following which would download the appropriate metadata # se <- tximeta(coldata) # for this example, we instead point to a local path where the GTF can be found # by making a linkedTxome: indexDir <- file.path(dir, "Dm.BDGP6.22.98_salmon-0.14.1") fastaFTP <- c("ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/cdna/Drosophila_melanogaster.BDGP6.22.cdna.all.fa.gz", "ftp://ftp.ensembl.org/pub/release-98/fasta/drosophila_melanogaster/ncrna/Drosophila_melanogaster.BDGP6.22.ncrna.fa.gz") gtfPath <- file.path(dir, "Drosophila_melanogaster.BDGP6.22.98.gtf.gz") makeLinkedTxome(indexDir=indexDir, source="LocalEnsembl", organism="Drosophila melanogaster", release="98", genome="BDGP6.22", fasta=fastaFTP, gtf=gtfPath, write=FALSE) se <- tximeta(coldata) # to clear the entire linkedTxome table # (don't run unless you want to clear this table!) # bfcloc <- getTximetaBFC() # bfc <- BiocFileCache(bfcloc) # bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)