Tximeta: transcript quantification import with automatic metadata
Michael I. Love, Charlotte Soneson, Peter F. Hickey, Rob Patro
01/29/2024
Abstract
Tximeta performs numerous annotation and metadata gathering tasks on behalf of users during the import of transcript quantifications from Salmon, alevin, or piscem-infer into R/Bioconductor. Metadata and transcript ranges are added automatically, facilitating genomic analyses and assisting in computational reproducibility.
Introduction
The tximeta package (Love et al. 2020) extends the tximport package (Soneson, Love, and Robinson 2015) for import of transcript-level quantification data into R/Bioconductor. It automatically adds annotation metadata when the RNA-seq data has been quantified with Salmon (Patro et al. 2017) or piscem-infer, or the scRNA-seq data quantified with alevin (Srivastava et al. 2019). To our knowledge, tximeta is the only package for RNA-seq data import that can automatically identify and attach transcriptome metadata based on the unique sequence of the reference transcripts. For more details on these packages – including the motivation for tximeta and description of similar work – consult the References below.
Note: tximeta requires that the entire output of Salmon / piscem-infer / alevin is present and unmodified in order to identify the provenance of the reference transcripts. In general, it’s a good idea to not modify or re-arrange the output directory of bioinformatic software as other downstream software rely on and assume a consistent directory structure. For sharing multiple samples, one can use, for example, tar -czf to bundle up a set of Salmon output directories, or to bundle one alevin output directory. For tips on using tximeta with other quantifiers see the other quantifiers section below.
Tximeta import starts with sample table
The first step using tximeta is to read in the sample table, which will become the column data, colData, of the final object, a SummarizedExperiment. The sample table should contain all the information we need to identify the Salmon quantification directories. For alevin quantification, one should point to the quants_mat.gz file that contains the counts for all of the cells (also, in order to tximeta to work with alevin quantification, it requires that alevin was run using gene IDs in the tgMap step and not gene symbols).
Here we will use a Salmon quantification file in the tximportData package to demonstrate the usage of tximeta. We do not have a sample table, so we construct one in R. It is recommended to keep a sample table as a CSV or TSV file while working on an RNA-seq project with multiple samples.
dir <- system.file("extdata/salmon_dm", package="tximportData")
files <- file.path(dir, "SRR1197474", "quant.sf")
file.exists(files)## [1] TRUE## files
## 1 /home/biocbuild/bbs-3.18-bioc/R/site-library/tximportData/extdata/salmon_dm/SRR1197474/quant.sf
## names condition
## 1 SRR1197474 Atximeta expects at least two columns in coldata:
files- a pointer to thequant.sffilesnames- the unique names that should be used to identify samples
Running tximeta
Normally, we would just run tximeta like so:
However, to avoid downloading remote GTF files during this vignette, we will point to a GTF file saved locally (in the tximportData package). We link the transcriptome of the Salmon index to its locally saved GTF. The standard recommended usage of tximeta would be the code chunk above, or to specify a remote GTF source, not a local one. This following code is therefore not recommended for a typically workflow, but is particular to the vignette code.
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")
suppressPackageStartupMessages(library(tximeta))
makeLinkedTxome(indexDir=indexDir,
source="LocalEnsembl",
organism="Drosophila melanogaster",
release="98",
genome="BDGP6.22",
fasta=fastaFTP,
gtf=gtfPath,
write=FALSE)## saving linkedTxome in bfc (first time)## importing quantifications## reading in files with read_tsv## 1
## found matching linked transcriptome:
## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
## building TxDb with 'GenomicFeatures' package
## Import genomic features from the file as a GRanges object ... OK
## Prepare the 'metadata' data frame ... OK
## Make the TxDb object ... OK
## generating transcript ranges## Warning in checkAssays2Txps(assays, txps):
##
## Warning: the annotation is missing some transcripts that were quantified.
## 5 out of 33706 txps were missing from GTF/GFF but were in the indexed FASTA.
## (This occurs sometimes with Ensembl txps on haplotype chromosomes.)
## In order to build a ranged SummarizedExperiment, these txps were removed.
## To keep these txps, and to skip adding ranges, use skipMeta=TRUE
##
## Example missing txps: [FBtr0307759, FBtr0084079, FBtr0084080, ...]What happened?
tximeta recognized the hashed checksum of the transcriptome that the files were quantified against, it accessed the GTF file of the transcriptome source, found and attached the transcript ranges, and added the appropriate transcriptome and genome metadata. A remote GTF is only downloaded once, and a local or remote GTF is only parsed to build a TxDb or EnsDb once: if tximeta recognizes that it has seen this Salmon index before, it will use a cached version of the metadata and transcript ranges.
Note the warning above that 5 of the transcripts are missing from the GTF file and so are dropped from the final output. This is a problem coming from the annotation source, and not easily avoided by tximeta.
TxDb, EnsDb, and AnnotationHub
tximeta makes use of Bioconductor packages for storing transcript databases as TxDb or EnsDb objects, which both are connected by default to sqlite backends. For GENCODE and RefSeq GTF files, tximeta uses the GenomicFeatures package (Lawrence 2013) to parse the GTF and build a TxDb. For Ensembl GTF files, tximeta will first attempt to obtain the correct EnsDb object using AnnotationHub. The ensembldb package (Rainer, Gatto, and Weichenberger 2019) contains classes and methods for extracting relevant data from Ensembl files. If the EnsDb has already been made available on AnnotationHub, tximeta will download the database directly, which saves the user time parsing the GTF into a database (to avoid this, set useHub=FALSE). If the relevant EnsDb is not available on AnnotationHub, tximeta will build an EnsDb using ensembldb after downloading the GTF file. Again, the download/construction of a transcript database occurs only once, and upon subsequent usage of tximeta functions, the cached version will be used.
Pre-computed checksums
We plan to support a wide variety of sources and organisms for transcriptomes with pre-computed checksums, though for now the software focuses on predominantly human and mouse transcriptomes
The following checksums are supported in this version of tximeta:
| source | organism | releases |
|---|---|---|
| GENCODE | Homo sapiens | 23-45 |
| GENCODE | Mus musculus | M6-M34 |
| Ensembl | Homo sapiens | 76-111 |
| Ensembl | Mus musculus | 76-111 |
| Ensembl | Drosophila melanogaster | 79-111 |
| RefSeq | Homo sapiens | p1-p13 |
| RefSeq | Mus musculus | p2-p6 |
For Ensembl transcriptomes, we support the combined protein coding (cDNA) and non-coding (ncRNA) sequences, as well as the protein coding alone (although the former approach combining coding and non-coding transcripts is recommended for more accurate quantification).
tximeta also has functions to support linked transcriptomes, where one or more sources for transcript sequences have been combined or filtered. See the Linked transcriptome section below for a demonstration. (The makeLinkedTxome function was used above to avoid downloading the GTF during the vignette building process.)
SummarizedExperiment output
We have our coldata from before. Note that we’ve removed files.
## DataFrame with 1 row and 2 columns
## names condition
## <character> <character>
## SRR1197474 SRR1197474 AHere we show the three matrices that were imported.
## [1] "counts" "abundance" "length"If there were inferential replicates (Gibbs samples or bootstrap samples), these would be imported as additional assays named "infRep1", "infRep2", …
tximeta has imported the correct ranges for the transcripts:
## GRanges object with 33701 ranges and 3 metadata columns:
## seqnames ranges strand | tx_id gene_id
## <Rle> <IRanges> <Rle> | <integer> <CharacterList>
## FBtr0070129 X 656673-657899 + | 28756 FBgn0025637
## FBtr0070126 X 656356-657899 + | 28752 FBgn0025637
## FBtr0070128 X 656673-657899 + | 28755 FBgn0025637
## FBtr0070124 X 656114-657899 + | 28750 FBgn0025637
## FBtr0070127 X 656356-657899 + | 28753 FBgn0025637
## ... ... ... ... . ... ...
## FBtr0114299 2R 21325218-21325323 + | 9300 FBgn0086023
## FBtr0113582 3R 5598638-5598777 - | 24474 FBgn0082989
## FBtr0091635 3L 1488906-1489045 + | 13780 FBgn0086670
## FBtr0113599 3L 261803-261953 - | 16973 FBgn0083014
## FBtr0113600 3L 831870-832008 - | 17070 FBgn0083057
## tx_name
## <character>
## FBtr0070129 FBtr0070129
## FBtr0070126 FBtr0070126
## FBtr0070128 FBtr0070128
## FBtr0070124 FBtr0070124
## FBtr0070127 FBtr0070127
## ... ...
## FBtr0114299 FBtr0114299
## FBtr0113582 FBtr0113582
## FBtr0091635 FBtr0091635
## FBtr0113599 FBtr0113599
## FBtr0113600 FBtr0113600
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsWe have appropriate genome information, which prevents us from making bioinformatic mistakes:
## Seqinfo object with 25 sequences from an unspecified genome; no seqlengths:
## seqnames seqlengths isCircular genome
## 2L <NA> <NA> <NA>
## 2R <NA> <NA> <NA>
## 3L <NA> <NA> <NA>
## 3R <NA> <NA> <NA>
## 4 <NA> <NA> <NA>
## ... ... ... ...
## 211000022280481 <NA> <NA> <NA>
## 211000022280494 <NA> <NA> <NA>
## 211000022280703 <NA> <NA> <NA>
## mitochondrion_genome <NA> <NA> <NA>
## rDNA <NA> <NA> <NA>Retrieve the transcript database
The se object has associated metadata that allows tximeta to link to locally stored cached databases and other Bioconductor objects. In further sections, we will show examples functions that leverage this databases for adding exon information, summarize transcript-level data to the gene level, or add identifiers. However, first we mention that the user can easily access the cached database with the following helper function. In this case, tximeta has an associated EnsDb object that we can retrieve and use in our R session:
## loading existing TxDb created: 2024-01-30 00:41:46## Loading required package: GenomicFeatures## Loading required package: AnnotationDbi## [1] "TxDb"
## attr(,"package")
## [1] "GenomicFeatures"The database returned by retrieveDb is either a TxDb in the case of GENCODE or RefSeq GTF annotation file, or an EnsDb in the case of an Ensembl GTF annotation file. For further use of these two database objects, consult the GenomicFeatures vignettes and the ensembldb vignettes, respectively (both Bioconductor packages).
Add exons per transcript
Because the SummarizedExperiment maintains all the metadata of its creation, it also keeps a pointer to the necessary database for pulling out additional information, as demonstrated in the following sections.
If necessary, the tximeta package can pull down the remote source to build a TxDb, but given that we’ve already built a TxDb once, it simply loads the cached version. In order to remove the cached TxDb and regenerate, one can remove the relevant entry from the tximeta file cache that resides at the location given by getTximetaBFC().
The se object created by tximeta, has the start, end, and strand information for each transcript. Here, we swap out the transcript GRanges for exons-by-transcript GRangesList (it is a list of GRanges, where each element of the list gives the exons for a particular transcript).
## loading existing TxDb created: 2024-01-30 00:41:46## generating exon ranges## GRanges object with 2 ranges and 3 metadata columns:
## seqnames ranges strand | exon_id exon_name exon_rank
## <Rle> <IRanges> <Rle> | <integer> <character> <integer>
## [1] X 656673-656740 + | 72949 FBtr0070129-E1 1
## [2] X 657099-657899 + | 72952 FBtr0070129-E2 2
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsAs with the transcript ranges, the exon ranges will be generated once and cached locally. As it takes a non-negligible amount of time to generate the exon-by-transcript GRangesList, this local caching offers substantial time savings for repeated usage of addExons with the same transcriptome.
We have implemented addExons to work only on the transcript-level SummarizedExperiment object. We provide some motivation for this choice in ?addExons. Briefly, if it is desired to know the exons associated with a particular gene, we feel that it makes more sense to pull out the relevant set of exons-by-transcript for the transcripts for this gene, rather than losing the hierarchical structure (exons to transcripts to genes) that would occur with a GRangesList of exons grouped per gene.
Easy summarization to gene-level
Likewise, the tximeta package can make use of the cached TxDb database for the purpose of summarizing transcript-level quantifications and bias corrections to the gene-level. After summarization, the rowRanges reflect the start and end position of the gene, which in Bioconductor are defined by the leftmost and rightmost genomic coordinates of all the transcripts. As with the transcript and exons, the gene ranges are cached locally for repeated usage. The transcript IDs are stored as a CharacterList column tx_ids.
## loading existing TxDb created: 2024-01-30 00:41:46## obtaining transcript-to-gene mapping from database## generating gene ranges## gene ranges assigned by total range of isoforms, see `assignRanges`## summarizing abundance## summarizing counts## summarizing length## GRanges object with 17208 ranges and 2 metadata columns:
## seqnames ranges strand | gene_id
## <Rle> <IRanges> <Rle> | <character>
## FBgn0000003 3R 6822498-6822796 + | FBgn0000003
## FBgn0000008 2R 22136968-22172834 + | FBgn0000008
## FBgn0000014 3R 16807214-16830049 - | FBgn0000014
## FBgn0000015 3R 16927212-16972236 - | FBgn0000015
## FBgn0000017 3L 16615866-16647882 - | FBgn0000017
## ... ... ... ... . ...
## FBgn0286199 3R 24279572-24281576 + | FBgn0286199
## FBgn0286203 2R 5413744-5456095 + | FBgn0286203
## FBgn0286204 3R 8950246-8963037 - | FBgn0286204
## FBgn0286213 3L 13023352-13024762 + | FBgn0286213
## FBgn0286222 X 6678424-6681845 + | FBgn0286222
## tx_ids
## <CharacterList>
## FBgn0000003 FBtr0081624
## FBgn0000008 FBtr0071763,FBtr0100521,FBtr0342981,...
## FBgn0000014 FBtr0306337,FBtr0083388,FBtr0083387,...
## FBgn0000015 FBtr0415463,FBtr0415464,FBtr0083385,...
## FBgn0000017 FBtr0112790,FBtr0345369,FBtr0075357,...
## ... ...
## FBgn0286199 FBtr0084600
## FBgn0286203 FBtr0299918,FBtr0299920,FBtr0299921,...
## FBgn0286204 FBtr0082014,FBtr0334329
## FBgn0286213 FBtr0075878
## FBgn0286222 FBtr0070953,FBtr0070954
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengthsAssign ranges by abundance
We also offer a new type of range assignment, based on the most abundant isoform rather than the leftmost to rightmost coordinate. See the assignRanges argument of ?summarizeToGene. Using the most abundant isoform arguably will reflect more accurate genomic distances than the default option.
For more explanation about why this may be a better choice, see the following tutorial chapter:
https://tidyomics.github.io/tidy-ranges-tutorial/gene-ranges-in-tximeta.html
In the below diagram, the pink feature is the set of all exons belonging to any isoform of the gene, such that the TSS is on the right side of this minus strand feature. However, the blue feature is the most abundant isoform (the brown features are the next most abundant isoforms). The pink feature is therefore not a good representation for the locus.
Add different identifiers
We would like to add support to easily map transcript or gene identifiers from one annotation to another. This is just a prototype function, but we show how we can easily add alternate IDs given that we know the organism and the source of the transcriptome. (This function currently only works for GENCODE and Ensembl gene or transcript IDs but could be extended to work for arbitrary sources.)
## ## mapping to new IDs using org.Dm.eg.db
## if all matching IDs are desired, and '1:many mappings' are reported,
## set multiVals='list' to obtain all the matching IDs## 'select()' returned 1:many mapping between keys and columns## DataFrame with 17208 rows and 3 columns
## gene_id tx_ids REFSEQ
## <character> <CharacterList> <character>
## FBgn0000003 FBgn0000003 FBtr0081624 NR_001992
## FBgn0000008 FBgn0000008 FBtr0071763,FBtr0100521,FBtr0342981,... NM_001014543
## FBgn0000014 FBgn0000014 FBtr0306337,FBtr0083388,FBtr0083387,... NM_001170161
## FBgn0000015 FBgn0000015 FBtr0415463,FBtr0415464,FBtr0083385,... NM_001275719
## FBgn0000017 FBgn0000017 FBtr0112790,FBtr0345369,FBtr0075357,... NM_001104153
## ... ... ... ...
## FBgn0286199 FBgn0286199 FBtr0084600 NM_142982
## FBgn0286203 FBgn0286203 FBtr0299918,FBtr0299920,FBtr0299921,... NM_001144134
## FBgn0286204 FBgn0286204 FBtr0082014,FBtr0334329 NM_001275464
## FBgn0286213 FBgn0286213 FBtr0075878 NM_168534
## FBgn0286222 FBgn0286222 FBtr0070953,FBtr0070954 NM_132111Differential expression analysis
The following code chunk demonstrates how to build a DESeqDataSet and begin a differential expression analysis.
suppressPackageStartupMessages(library(DESeq2))
# here there is a single sample so we use ~1.
# expect a warning that there is only a single sample...
suppressWarnings({dds <- DESeqDataSet(gse, ~1)})## using counts and average transcript lengths from tximetaThe Swish method in the fishpond package directly works with the SummarizedExperiment output from tximeta, and can perform differential analysis on transcript expression taking into account inferential replicates, e.g. bootstrap or Gibbs samples, which are imported and arranged by tximeta if these were generated during quantification.
library(fishpond)
y <- se
# y <- scaleInfReps(y)
# y <- labelKeep(y)
# y <- swish(y, x="condition")
# ... see Swish vignette in fishpond packageWe have a convenient wrapper function that will build a DGEList object for use with edgeR.
suppressPackageStartupMessages(library(edgeR))
y <- makeDGEList(gse)
# ... see edgeR User's Guide for further stepsThe following code chunk demonstrates the code inside of the above wrapper function, and produces the same output.
cts <- assays(gse)[["counts"]]
normMat <- assays(gse)[["length"]]
normMat <- normMat / exp(rowMeans(log(normMat)))
o <- log(calcNormFactors(cts/normMat)) + log(colSums(cts/normMat))
y <- DGEList(cts)
y <- scaleOffset(y, t(t(log(normMat)) + o))
# ... see edgeR User's Guide for further stepsThe following code chunk demonstrates how one could use the Swish method in the fishpond Bioconductor package. Here we use the transcript-level object se. This dataset only has a single sample and no inferential replicates, but the analysis would begin with such code. See the Swish vignette in the fishpond package for a complete example:
y <- se # rename the object to 'y'
library(fishpond)
# if inferential replicates existed in the data,
# analysis would begin with:
#
# y <- scaleInfReps(y)
# ... see Swish vignette in the fishpond packageFor limma with voom transformation we recommend, as in the tximport vignette to generate counts-from-abundance instead of providing an offset for average transcript length.
## loading existing TxDb created: 2024-01-30 00:41:46## obtaining transcript-to-gene mapping from database## loading existing gene ranges created: 2024-01-30 00:41:51## gene ranges assigned by total range of isoforms, see `assignRanges`## summarizing abundance## summarizing counts## summarizing lengthAbove we generated counts-from-abundance when calling summarizeToGene. The counts-from-abundance status is then stored in the metadata:
## [1] "lengthScaledTPM"Additional metadata
The following information is attached to the SummarizedExperiment by tximeta:
## [1] "tximetaInfo" "quantInfo" "countsFromAbundance"
## [4] "level" "txomeInfo" "txdbInfo"## List of 31
## $ salmon_version : chr "0.14.1"
## $ samp_type : chr "none"
## $ opt_type : chr "vb"
## $ quant_errors :List of 1
## ..$ : list()
## $ num_libraries : int 1
## $ library_types : chr "ISR"
## $ frag_dist_length : int 1001
## $ seq_bias_correct : logi FALSE
## $ gc_bias_correct : logi TRUE
## $ num_bias_bins : int 4096
## $ mapping_type : chr "mapping"
## $ num_valid_targets : int 33706
## $ num_decoy_targets : int 0
## $ num_eq_classes : int 70718
## $ serialized_eq_classes : logi FALSE
## $ length_classes : int [1:5, 1] 867 1533 2379 3854 71382
## $ index_seq_hash : chr "7ba5e9597796ea86cf11ccf6635ca88fbc37c2848d38083c23986aa2c6a21eae"
## $ index_name_hash : chr "b6426061057bba9b7afb4dc76fa68238414cf35b4190c95ca6fc44280d4ca87c"
## $ index_seq_hash512 : chr "05f111abcda1efd2e489ace6324128cdaaa311712a28ed716d957fdfd8706ec41ca9177ebf12f54e99c2a89582d06f31c5e09dc1dce2d13"| __truncated__
## $ index_name_hash512 : chr "ccdf58f23e48c8c53cd122b5f5990b5adce9fec87ddf8bd88153afbe93296d87b818fba89d12dbc20c882f7d98353840394c5040fea7432"| __truncated__
## $ num_bootstraps : int 0
## $ num_processed : int 42422337
## $ num_mapped : int 34098209
## $ num_decoy_fragments : int 0
## $ num_dovetail_fragments : int 2048810
## $ num_fragments_filtered_vm : int 989383
## $ num_alignments_below_threshold_for_mapped_fragments_vm: int 267540
## $ percent_mapped : num 80.4
## $ call : chr "quant"
## $ start_time : chr "Sat Oct 12 13:55:01 2019"
## $ end_time : chr "Sat Oct 12 14:08:11 2019"## List of 9
## $ index : chr "Dm.BDGP6.22.98_salmon-0.14.1"
## $ source : chr "LocalEnsembl"
## $ organism : chr "Drosophila melanogaster"
## $ release : chr "98"
## $ genome : chr "BDGP6.22"
## $ fasta :List of 1
## ..$ : chr [1:2] "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"
## $ gtf : chr "/home/biocbuild/bbs-3.18-bioc/R/site-library/tximportData/extdata/salmon_dm/Drosophila_melanogaster.BDGP6.22.98.gtf.gz"
## $ sha256 : chr "7ba5e9597796ea86cf11ccf6635ca88fbc37c2848d38083c23986aa2c6a21eae"
## $ linkedTxome: logi TRUE## List of 3
## $ version :Classes 'package_version', 'numeric_version' hidden list of 1
## ..$ : int [1:3] 1 20 3
## $ type : chr "salmon"
## $ importTime: POSIXct[1:1], format: "2024-01-29 19:41:34"## Named chr [1:13] "TxDb" "GenomicFeatures" ...
## - attr(*, "names")= chr [1:13] "Db type" "Supporting package" "Data source" "Organism" ...Errors connecting to a database
tximeta makes use of BiocFileCache to store transcript and other databases, so saving the relevant databases in a centralized location used by other Bioconductor packages as well. It is possible that an error can occur in connecting to these databases, either if the files were accidentally removed from the file system, or if there was an error generating or writing the database to the cache location. In each of these cases, it is easy to remove the entry in the BiocFileCache so that tximeta will know to regenerate the transcript database or any other missing database.
If you have used the default cache location, then you can obtain access to your BiocFileCache with:
## Loading required package: dbplyrOtherwise, you can recall your particular tximeta cache location with getTximetaBFC().
You can then inspect the entries in your BiocFileCache using bfcinfo and remove the entry associated with the missing database with bfcremove. See the BiocFileCache vignette for more details on finding and removing entries from a BiocFileCache.
Note that there may be many entries in the BiocFileCache location, including .sqlite database files and serialized .rds files. You should only remove the entry associated with the missing database, e.g. if R gave an error when trying to connect to the TxDb associated with GENCODE v99 human transcripts, you should look for the rid of the entry associated with the human v99 GTF from GENCODE.
What if checksum isn’t known?
tximeta automatically imports relevant metadata when the transcriptome matches a known source – known in the sense that it is in the set of pre-computed hashed checksums in tximeta (GENCODE, Ensembl, and RefSeq for human and mouse). tximeta also facilitates the linking of transcriptomes used in building the Salmon index with relevant public sources, in the case that these are not part of this pre-computed set known to tximeta. The linking of the transcriptome source with the quantification files is important in the case that the transcript sequence no longer matches a known source (uniquely combined or filtered FASTA files), or if the source is not known to tximeta. Combinations of coding and non-coding human, mouse, and fruit fly Ensembl transcripts should be automatically recognized by tximeta and does not require making a linkedTxome. As the package is further developed, we plan to roll out support for all common transcriptomes, from all sources.
Note: if you are using Salmon in alignment mode, then there is no Salmon index, and without the Salmon index, there is no checksum. We don’t have a perfect solution for this yet, but you can still summarize transcript counts to gene with a tx2gene table that you construct manually (see tximport vignette for example code). Just specify the arguments, skipMeta=TRUE, txOut=FALSE, tx2gene=tx2gene, when calling tximeta and it will perform summarization to gene level as in tximport.
We now demonstrate how to make a linkedTxome and how to share and load a linkedTxome. We point to a Salmon quantification file which was quantified against a transcriptome that included the coding and non-coding Drosophila melanogaster transcripts, as well as an artificial transcript of 960 bp (for demonstration purposes only).
## [1] TRUETrying to import the files gives a message that tximeta couldn’t find a matching transcriptome, so it returns an non-ranged SummarizedExperiment.
## importing quantifications## reading in files with read_tsv## 1
## couldn't find matching transcriptome, returning non-ranged SummarizedExperimentLinked transcriptomes
If the transcriptome used to generate the Salmon index does not match any transcriptomes from known sources (e.g. from combining or filtering known transcriptome files), there is not much that can be done to automatically populate the metadata during quantification import. However, we can facilitate the following two cases:
- the transcriptome was created locally and has been linked to its public source(s)
- the transcriptome was produced by another group, and they have produced and shared a file that links the transcriptome to public source(s)
tximeta offers functionality to assist reproducible analysis in both of these cases.
To make this quantification reproducible, we make a linkedTxome which records key information about the sources of the transcript FASTA files, and the location of the relevant GTF file. It also records the checksum of the transcriptome that was computed by Salmon during the index step.
Source: when creating the linkedTxome one must specify the source of the transcriptome. See ?linkedTxome for a note on the implications of this text string. For canonical GENCODE or Ensembl transcriptomes, one can use "GENCODE" or "Ensembl", but for modified or otherwise any transcriptomes defined by a local database, it is recommended to use a different string, "LocalGENCODE" or `“LocalEnsembl”, which will avoid tximeta pulling canonical GENCODE or Ensembl resources from AnnotationHub.
Multiple GTF/GFF files: linkedTxome and tximeta do not currently support multiple GTF/GFF files, which is a more complicated case than multiple FASTA, which is supported. Currently, we recommend that users should add or combine GTF/GFF files themselves to create a single GTF/GFF file that contains all features used in quantification, and then upload such a file to Zenodo, which can then be linked as shown below. Feel free to contact the developers on the Bioconductor support site or GitHub Issue page for further details or feature requests.
Stringtie: A special note for building on top of Stringtie-generated transcripts: it is a good idea to change gene identifiers, to not include a period ., as the period will later be used to separate transcript versions from gene identifiers. This can be done before building the Salmon index, by changing periods in the gene identifier to an underscore. See this GitHub issue for details.
By default, linkedTxome will write out a JSON file which can be shared with others, linking the checksum of the index with the other metadata, including FASTA and GTF sources. By default, it will write out to a file with the same name as the indexDir, but with a .json extension added. This can be prevented with write=FALSE, and the file location can be changed with jsonFile.
First we specify the path where the Salmon index is located.
Typically you would not use system.file and file.path to locate this directory, but simply define indexDir to be the path of the Salmon directory on your machine. Here we use system.file and file.path because we have included parts of a Salmon index directory in the tximeta package itself for demonstration of functionality in this vignette.
Now we provide the location of the FASTA files and the GTF file for this transcriptome.
Note: the basename for the GTF file is used as a unique identifier for the cached versions of the TxDb and the transcript ranges, which are stored on the user’s behalf via BiocFileCache. This is not an issue, as GENCODE, Ensembl, and RefSeq all provide GTF files which are uniquely identified by their filename, e.g. Drosophila_melanogaster.BDGP6.22.98.gtf.gz.
The recommended usage of tximeta would be to specify a remote GTF source, as seen in the commented-out line below:
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")
#gtfFTP <- "ftp://path/to/custom/Drosophila_melanogaster.BDGP6.22.98.plus.gtf.gz"Instead of the above commented-out FTP location for the GTF file, we specify a location within an R package. This step is just to avoid downloading from a remote FTP during vignette building. This use of file.path to point to a file in an R package is specific to this vignette and should not be used in a typical workflow. The following GTF file is a modified version of the release 98 from Ensembl, which includes description of a one transcript, one exon artificial gene which was inserted into the transcriptome (for demonstration purposes only).
Finally, we create a linkedTxome. In this vignette, we point to a temporary directory for the JSON file, but a more typical workflow would write the JSON file to the same location as the Salmon index by not specifying jsonFile.
makeLinkedTxome performs two operation: (1) it creates a new entry in an internal table that links the transcriptome used in the Salmon index to its sources, and (2) it creates a JSON file such that this linkedTxome can be shared.
tmp <- tempdir()
jsonFile <- file.path(tmp, paste0(basename(indexDir), ".json"))
makeLinkedTxome(indexDir=indexDir,
source="LocalEnsembl", organism="Drosophila melanogaster",
release="98", genome="BDGP6.22",
fasta=fastaFTP, gtf=gtfPath,
jsonFile=jsonFile)## writing linkedTxome to /tmp/RtmpSJcbCB/Dm.BDGP6.22.98.plus_salmon-0.14.1.json## saving linkedTxome in bfcAfter running makeLinkedTxome, the connection between this Salmon index (and its checksum) with the sources is saved for persistent usage. Note that because we added a single transcript of 960bp to the FASTA file used for quantification, tximeta could tell that this was not quantified against release 98 of the Ensembl transcripts for Drosophila melanogaster. Only when the correct set of transcripts were specified does tximeta recognize and import the correct metadata.
With use of tximeta and a linkedTxome, the software figures out if the remote GTF has been accessed and compiled into a TxDb before, and on future calls, it will simply load the pre-computed metadata and transcript ranges.
Note the warning that 5 of the transcripts are missing from the GTF file and so are dropped from the final output. This is a problem coming from the annotation source, and not easily avoided by tximeta.
## importing quantifications## reading in files with read_tsv## 1
## found matching linked transcriptome:
## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
## building TxDb with 'GenomicFeatures' package
## Import genomic features from the file as a GRanges object ... OK
## Prepare the 'metadata' data frame ... OK
## Make the TxDb object ... OK
## generating transcript ranges## Warning in checkAssays2Txps(assays, txps):
##
## Warning: the annotation is missing some transcripts that were quantified.
## 5 out of 33707 txps were missing from GTF/GFF but were in the indexed FASTA.
## (This occurs sometimes with Ensembl txps on haplotype chromosomes.)
## In order to build a ranged SummarizedExperiment, these txps were removed.
## To keep these txps, and to skip adding ranges, use skipMeta=TRUE
##
## Example missing txps: [FBtr0307759, FBtr0084079, FBtr0084080, ...]We can see that the appropriate metadata and transcript ranges are attached.
## GRanges object with 33702 ranges and 3 metadata columns:
## seqnames ranges strand | tx_id gene_id
## <Rle> <IRanges> <Rle> | <integer> <CharacterList>
## Newgene 3R 1-960 + | 20219 Newgene
## FBtr0070129 X 656673-657899 + | 28757 FBgn0025637
## FBtr0070126 X 656356-657899 + | 28753 FBgn0025637
## FBtr0070128 X 656673-657899 + | 28756 FBgn0025637
## FBtr0070124 X 656114-657899 + | 28751 FBgn0025637
## ... ... ... ... . ... ...
## FBtr0114299 2R 21325218-21325323 + | 9300 FBgn0086023
## FBtr0113582 3R 5598638-5598777 - | 24475 FBgn0082989
## FBtr0091635 3L 1488906-1489045 + | 13780 FBgn0086670
## FBtr0113599 3L 261803-261953 - | 16973 FBgn0083014
## FBtr0113600 3L 831870-832008 - | 17070 FBgn0083057
## tx_name
## <character>
## Newgene Newgene
## FBtr0070129 FBtr0070129
## FBtr0070126 FBtr0070126
## FBtr0070128 FBtr0070128
## FBtr0070124 FBtr0070124
## ... ...
## FBtr0114299 FBtr0114299
## FBtr0113582 FBtr0113582
## FBtr0091635 FBtr0091635
## FBtr0113599 FBtr0113599
## FBtr0113600 FBtr0113600
## -------
## seqinfo: 25 sequences from an unspecified genome; no seqlengths## Seqinfo object with 25 sequences from an unspecified genome; no seqlengths:
## seqnames seqlengths isCircular genome
## 2L <NA> <NA> <NA>
## 2R <NA> <NA> <NA>
## 3L <NA> <NA> <NA>
## 3R <NA> <NA> <NA>
## 4 <NA> <NA> <NA>
## ... ... ... ...
## 211000022280481 <NA> <NA> <NA>
## 211000022280494 <NA> <NA> <NA>
## 211000022280703 <NA> <NA> <NA>
## mitochondrion_genome <NA> <NA> <NA>
## rDNA <NA> <NA> <NA>Clear linkedTxomes
The following code removes the entire table with information about the linkedTxomes. This is just for demonstration, so that we can show how to load a JSON file below.
Note: Running this code will clear any information about linkedTxomes. Don’t run this unless you really want to clear this table!
library(BiocFileCache)
if (interactive()) {
bfcloc <- getTximetaBFC()
} else {
bfcloc <- tempdir()
}
bfc <- BiocFileCache(bfcloc)
bfcinfo(bfc)## # A tibble: 7 × 10
## rid rname create_time access_time rpath rtype fpath last_modified_time etag
## <chr> <chr> <chr> <chr> <chr> <chr> <chr> <dbl> <chr>
## 1 BFC1 link… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 2 BFC2 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 3 BFC3 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 4 BFC4 exon… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 5 BFC5 gene… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 6 BFC6 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 7 BFC7 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## # ℹ 1 more variable: expires <dbl>## # A tibble: 6 × 10
## rid rname create_time access_time rpath rtype fpath last_modified_time etag
## <chr> <chr> <chr> <chr> <chr> <chr> <chr> <dbl> <chr>
## 1 BFC2 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 2 BFC3 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 3 BFC4 exon… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 4 BFC5 gene… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 5 BFC6 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 6 BFC7 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## # ℹ 1 more variable: expires <dbl>Loading linkedTxome JSON files
If a collaborator or the Suppmentary Files for a publication shares a linkedTxome JSON file, we can likewise use tximeta to automatically assemble the relevant metadata and transcript ranges. This implies that the other person has used tximeta with the function makeLinkedTxome demonstrated above, pointing to their Salmon index and to the FASTA and GTF source(s).
We point to the JSON file and use loadLinkedTxome and then the relevant metadata is saved for persistent usage. In this case, we saved the JSON file in a temporary directory.
## saving linkedTxome in bfc (first time)Again, using tximeta figures out whether it needs to access the remote GTF or not, and assembles the appropriate object on the user’s behalf.
## importing quantifications## reading in files with read_tsv## 1
## found matching linked transcriptome:
## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
## loading existing TxDb created: 2024-01-30 00:42:06
## loading existing transcript ranges created: 2024-01-30 00:42:07## Warning in checkAssays2Txps(assays, txps):
##
## Warning: the annotation is missing some transcripts that were quantified.
## 5 out of 33707 txps were missing from GTF/GFF but were in the indexed FASTA.
## (This occurs sometimes with Ensembl txps on haplotype chromosomes.)
## In order to build a ranged SummarizedExperiment, these txps were removed.
## To keep these txps, and to skip adding ranges, use skipMeta=TRUE
##
## Example missing txps: [FBtr0307759, FBtr0084079, FBtr0084080, ...]Clear linkedTxomes again
Finally, we clear the linkedTxomes table again so that the above examples will work. This is just for the vignette code and not part of a typical workflow.
Note: Running this code will clear any information about linkedTxomes. Don’t run this unless you really want to clear this table!
if (interactive()) {
bfcloc <- getTximetaBFC()
} else {
bfcloc <- tempdir()
}
bfc <- BiocFileCache(bfcloc)
bfcinfo(bfc)## # A tibble: 7 × 10
## rid rname create_time access_time rpath rtype fpath last_modified_time etag
## <chr> <chr> <chr> <chr> <chr> <chr> <chr> <dbl> <chr>
## 1 BFC2 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 2 BFC3 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 3 BFC4 exon… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 4 BFC5 gene… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 5 BFC6 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 6 BFC7 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 7 BFC8 link… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## # ℹ 1 more variable: expires <dbl>## # A tibble: 6 × 10
## rid rname create_time access_time rpath rtype fpath last_modified_time etag
## <chr> <chr> <chr> <chr> <chr> <chr> <chr> <dbl> <chr>
## 1 BFC2 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 2 BFC3 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 3 BFC4 exon… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 4 BFC5 gene… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 5 BFC6 Dros… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## 6 BFC7 txpR… 2024-01-30… 2024-01-30… /tmp… rela… 7f62… NA <NA>
## # ℹ 1 more variable: expires <dbl>Other quantifiers
tximeta can import the output from any quantifiers that are supported by tximport, and if these are not Salmon, alevin, or Sailfish output, it will simply return a non-ranged SummarizedExperiment by default.
An alternative solution is to wrap other quantifiers in workflows that include metadata information JSON files along with each quantification file. One can place these files in aux_info/meta_info.json or any relative location specified by customMetaInfo, for example customMetaInfo="meta_info.json". This JSON file is located relative to the quantification file and should contain a tag index_seq_hash with an associated value of the SHA-256 hash of the reference transcripts. For computing the hash value of the reference transcripts, see the FastaDigest python package. The hash value used by Salmon is the SHA-256 hash value of the reference sequences stripped of the header lines, and concatenated together with the empty string (so only cDNA sequences combined without any new line characters). FastaDigest can be installed with pip install fasta_digest.
Automated analysis with ARMOR
This vignette described the use of tximeta to import quantification data into R/Bioconductor with automatic detection and addition of metadata. The SummarizedExperiment produced by tximeta can then be provided to downstream statistical analysis packages as described above. The tximeta package does not contain any functionality for automated differential analysis.
The ARMOR workflow does automate a variety of differential analyses, and make use of tximeta for creation of a SummarizedExperiment with attached annotation metadata. ARMOR stands for ``An Automated Reproducible MOdular Workflow for Preprocessing and Differential Analysis of RNA-seq Data’’ and is described in more detail in the article by Orjuela et al. (2019).
Default BiocFileCahce Caching Location Update
tximeta makes use of the default BiocFileCache location, unless otherwise specified by the user. As of BiocFileCache version > 1.15.1, the default caching location used by BiocFileCache has changed. In order to continue to use the same cache (without re-downloading files), please follow the steps in the BiocFileCache vignette, under the heading Default Caching Location Update.
Acknowledgments
The development of tximeta has benefited from suggestions from these and other individuals in the community:
- Vincent Carey
- Lori Shepherd
- Martin Morgan
- Koen Van den Berge
- Johannes Rainer
- James Ashmore
- Ben Johnson
- Tim Triche
- Kristoffer Vitting-Seerup
Session info
## Loading required package: usethis## ─ Session info ───────────────────────────────────────────────────────────────
## setting value
## version R version 4.3.2 Patched (2023-11-13 r85521)
## os Ubuntu 22.04.3 LTS
## system x86_64, linux-gnu
## ui X11
## language (EN)
## collate C
## ctype en_US.UTF-8
## tz America/New_York
## date 2024-01-29
## pandoc 2.7.3 @ /usr/bin/ (via rmarkdown)
##
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## rtracklayer 1.62.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## S4Arrays 1.2.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## S4Vectors * 0.40.2 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## sass 0.4.8 2023-12-06 [2] CRAN (R 4.3.2)
## scales 1.3.0 2023-11-28 [2] CRAN (R 4.3.2)
## sessioninfo 1.2.2 2021-12-06 [2] CRAN (R 4.3.2)
## shiny 1.8.0 2023-11-17 [2] CRAN (R 4.3.2)
## SingleCellExperiment 1.24.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## SparseArray 1.2.3 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## statmod 1.5.0 2023-01-06 [2] CRAN (R 4.3.2)
## stringi 1.8.3 2023-12-11 [2] CRAN (R 4.3.2)
## stringr 1.5.1 2023-11-14 [2] CRAN (R 4.3.2)
## SummarizedExperiment * 1.32.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## svMisc 1.2.3 2021-10-11 [2] CRAN (R 4.3.2)
## tibble 3.2.1 2023-03-20 [2] CRAN (R 4.3.2)
## tidyselect 1.2.0 2022-10-10 [2] CRAN (R 4.3.2)
## tximeta * 1.20.3 2024-01-29 [1] Bioconductor 3.18 (R 4.3.2)
## tximport 1.30.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## tzdb 0.4.0 2023-05-12 [2] CRAN (R 4.3.2)
## urlchecker 1.0.1 2021-11-30 [2] CRAN (R 4.3.2)
## usethis * 2.2.2 2023-07-06 [2] CRAN (R 4.3.2)
## utf8 1.2.4 2023-10-22 [2] CRAN (R 4.3.2)
## vctrs 0.6.5 2023-12-01 [2] CRAN (R 4.3.2)
## vroom 1.6.5 2023-12-05 [2] CRAN (R 4.3.2)
## withr 3.0.0 2024-01-16 [2] CRAN (R 4.3.2)
## xfun 0.41 2023-11-01 [2] CRAN (R 4.3.2)
## XML 3.99-0.16.1 2024-01-22 [2] CRAN (R 4.3.2)
## xml2 1.3.6 2023-12-04 [2] CRAN (R 4.3.2)
## xtable 1.8-4 2019-04-21 [2] CRAN (R 4.3.2)
## XVector 0.42.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
## yaml 2.3.8 2023-12-11 [2] CRAN (R 4.3.2)
## zlibbioc 1.48.0 2024-01-29 [2] Bioconductor 3.18 (R 4.3.2)
##
## [1] /tmp/RtmpDwpSMc/Rinst7b74e174b8cb2
## [2] /home/biocbuild/bbs-3.18-bioc/R/site-library
## [3] /home/biocbuild/bbs-3.18-bioc/R/library
##
## ──────────────────────────────────────────────────────────────────────────────References
Lawrence, Wolfgang AND Pagès, Michael AND Huber. 2013. “Software for Computing and Annotating Genomic Ranges.” PLOS Computational Biology 9 (8): 1–10.
Love, Michael I., Charlotte Soneson, Peter F. Hickey, Lisa K. Johnson, N. Tessa Pierce, Lori Shepherd, Martin Morgan, and Rob Patro. 2020. “Tximeta: Reference sequence checksums for provenance identification in RNA-seq.” PLOS Computational Biology 16 (2): e1007664. https://doi.org/10.1371/journal.pcbi.1007664.
Orjuela, Stephany, Ruizhu Huang, Katharina M. Hembach, Mark D. Robinson, and Charlotte Soneson. 2019. “ARMOR: An Automated Reproducible MOdular Workflow for Preprocessing and Differential Analysis of RNA-seq Data.” G3: Genes, Genomes, Genetics.
Patro, Rob, Geet Duggal, Michael I. Love, Rafael A. Irizarry, and Carl Kingsford. 2017. “Salmon Provides Fast and Bias-Aware Quantification of Transcript Expression.” Nature Methods. https://doi.org/10.1038/nmeth.4197.
Rainer, Johannes, Laurent Gatto, and Christian X Weichenberger. 2019. “ensembldb: an R package to create and use Ensembl-based annotation resources.” Bioinformatics, January.
Soneson, Charlotte, Michael I. Love, and Mark Robinson. 2015. “Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences.” F1000Research 4 (1521). https://doi.org/10.12688/f1000research.7563.1.
Srivastava, Avi, Laraib Malik, Tom Sean Smith, Ian Sudbery, and Rob Patro. 2019. “Alevin efficiently estimates accurate gene abundances from dscRNA-seq data.” Genome Biology 20 (65). https://doi.org/10.1186/s13059-019-1670-y.