# Tximeta: transcript quantification import with automatic metadata

#### Michael I. Love, Charlotte Soneson, Peter F. Hickey, Rob Patro

#### 05/14/2024

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

    coldata <- data.frame(files, names="SRR1197474", condition="A", stringsAsFactors=FALSE)
    coldata

    ##                                                                                     files
    ## 1 F:/biocbuild/bbs-3.19-bioc/R/library/tximportData/extdata/salmon_dm/SRR1197474/quant.sf
    ##        names condition
    ## 1 SRR1197474         A

`tximeta` expects at least two columns in `coldata`:

1. `files` - a pointer to the `quant.sf` files
2. `names` - the unique names that should be used to identify samples

# Running tximeta

Normally, we would just run `tximeta` like so:

    library(tximeta)
    se <- tximeta(coldata)

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)

    library(tximeta)

    se <- tximeta(coldata)

    ## importing quantifications

    ## reading in files with read_tsv

    ## 1
    ## found matching linked transcriptome:
    ## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
    ## building TxDb with 'txdbmaker' 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 computed digest 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 digest is a small string of alphanumeric characters that uniquely identifies the collection of sequences that were used for quantification (it is the application of a hash function). We sometimes also call this value a “checksum” (in the tximeta paper).

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 txdbmaker 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 digests

We plan to support a wide variety of sources and organisms for transcriptomes with pre-computed digests, though for now the software focuses on predominantly human and mouse transcriptomes

The following digests are supported in this version of `tximeta`:

source | organism | releases
--- | --- | ---
GENCODE | Homo sapiens | 23-46
GENCODE | Mus musculus | M6-M35
Ensembl | Homo sapiens | 76-112
Ensembl | Mus musculus | 76-112
Ensembl | Drosophila melanogaster | 79-112
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`.

    suppressPackageStartupMessages(library(SummarizedExperiment))
    colData(se)

    ## DataFrame with 1 row and 2 columns
    ##                  names   condition
    ##            <character> <character>
    ## SRR1197474  SRR1197474           A

Here we show the three matrices that were imported.

    assayNames(se)

    ## [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:

    rowRanges(se)

    ## 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 seqlengths

We have appropriate genome information, which prevents us from making bioinformatic mistakes:

    seqinfo(se)

    ## 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:

    edb <- retrieveDb(se)

    ## loading existing TxDb created: 2024-05-15 02:32:43

    ## Loading required package: GenomicFeatures

    ## Loading required package: AnnotationDbi

    class(edb)

    ## [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).

    se.exons <- addExons(se)

    ## loading existing TxDb created: 2024-05-15 02:32:43

    ## generating exon ranges

    rowRanges(se.exons)[[1]]

    ## 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 seqlengths

As 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`.

    gse <- summarizeToGene(se)

    ## loading existing TxDb created: 2024-05-15 02:32:43

    ## 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

    rowRanges(gse)

    ## 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 seqlengths

## Assign 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.

    # unevaluated code chunk
    gse <- summarizeToGene(se, assignRanges="abundant")

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.)

    library(org.Dm.eg.db)

    ##

    gse <- addIds(gse, "REFSEQ", gene=TRUE)

    ## 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

    mcols(gse)

    ## 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_132111

# Differential 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 tximeta

    # ... see DESeq2 vignette

The 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 package

We 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 steps

The 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 steps

The 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 package

For 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.

    gse <- summarizeToGene(se, countsFromAbundance="lengthScaledTPM")

    ## loading existing TxDb created: 2024-05-15 02:32:43

    ## obtaining transcript-to-gene mapping from database

    ## loading existing gene ranges created: 2024-05-15 02:32:49

    ## gene ranges assigned by total range of isoforms, see `assignRanges`

    ## summarizing abundance

    ## summarizing counts

    ## summarizing length

    library(limma)
    y <- DGEList(assays(gse)[["counts"]])
    # see limma User's Guide for further steps

Above we generated counts-from-abundance when calling `summarizeToGene`. The counts-from-abundance status is then stored in the metadata:

    metadata(gse)$countsFromAbundance

    ## [1] "lengthScaledTPM"

# Additional metadata

The following information is attached to the SummarizedExperiment by `tximeta`:

    names(metadata(se))

    ## [1] "tximetaInfo"         "quantInfo"           "countsFromAbundance"
    ## [4] "level"               "txomeInfo"           "txdbInfo"

    str(metadata(se)[["quantInfo"]])

    ## 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"

    str(metadata(se)[["txomeInfo"]])

    ## 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 "F:/biocbuild/bbs-3.19-bioc/R/library/tximportData/extdata/salmon_dm/Drosophila_melanogaster.BDGP6.22.98.gtf.gz"
    ##  $ sha256     : chr "7ba5e9597796ea86cf11ccf6635ca88fbc37c2848d38083c23986aa2c6a21eae"
    ##  $ linkedTxome: logi TRUE

    str(metadata(se)[["tximetaInfo"]])

    ## List of 3
    ##  $ version   :Classes 'package_version', 'numeric_version'  hidden list of 1
    ##   ..$ : int [1:3] 1 22 1
    ##  $ type      : chr "salmon"
    ##  $ importTime: POSIXct[1:1], format: "2024-05-14 22:32:18"

    str(metadata(se)[["txdbInfo"]])

    ##  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:

    library(BiocFileCache)

    ## Loading required package: dbplyr

    bfc <- BiocFileCache()

Otherwise, 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 digest 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 digests 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 digest. 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).

    file <- file.path(dir, "SRR1197474.plus", "quant.sf")
    file.exists(file)

    ## [1] TRUE

    coldata <- data.frame(files=file, names="SRR1197474", sample="1",
                          stringsAsFactors=FALSE)

Trying to import the files gives a message that `tximeta` couldn’t find a matching transcriptome, so it returns an non-ranged SummarizedExperiment.

    se <- tximeta(coldata)

    ## importing quantifications

    ## reading in files with read_tsv

    ## 1
    ## couldn't find matching transcriptome, returning non-ranged SummarizedExperiment

# Linked 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:

1. the transcriptome was created locally and has been linked to its public source(s)
2. 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 digest 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 digest 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.

    indexDir <- file.path(dir, "Dm.BDGP6.22.98.plus_salmon-0.14.1")

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).

    gtfPath <- file.path(dir,"Drosophila_melanogaster.BDGP6.22.98.plus.gtf.gz")

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 F:\biocbuild\bbs-3.19-bioc\tmpdir\RtmpeqxpPS/Dm.BDGP6.22.98.plus_salmon-0.14.1.json

    ## saving linkedTxome in bfc

After running `makeLinkedTxome`, the connection between this Salmon index (and its digest) 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`.

    se <- tximeta(coldata)

    ## importing quantifications

    ## reading in files with read_tsv

    ## 1
    ## found matching linked transcriptome:
    ## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
    ## building TxDb with 'txdbmaker' 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.

    rowRanges(se)

    ## 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(se)

    ## 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… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 2 BFC2  Dros… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 3 BFC3  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 4 BFC4  exon… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 5 BFC5  gene… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 6 BFC6  Dros… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 7 BFC7  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## # ℹ 1 more variable: expires <dbl>

    bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)
    bfcinfo(bfc)

    ## # 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… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 2 BFC3  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 3 BFC4  exon… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 4 BFC5  gene… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 5 BFC6  Dros… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 6 BFC7  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 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.

    jsonFile <- file.path(tmp, paste0(basename(indexDir), ".json"))
    loadLinkedTxome(jsonFile)

    ## 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.

    se <- tximeta(coldata)

    ## importing quantifications

    ## reading in files with read_tsv

    ## 1
    ## found matching linked transcriptome:
    ## [ LocalEnsembl - Drosophila melanogaster - release 98 ]
    ## loading existing TxDb created: 2024-05-15 02:33:22
    ## loading existing transcript ranges created: 2024-05-15 02:33:23

    ## 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… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 2 BFC3  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 3 BFC4  exon… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 4 BFC5  gene… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 5 BFC6  Dros… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 6 BFC7  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 7 BFC8  link… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## # ℹ 1 more variable: expires <dbl>

    bfcremove(bfc, bfcquery(bfc, "linkedTxomeTbl")$rid)
    bfcinfo(bfc)

    ## # 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… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 2 BFC3  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 3 BFC4  exon… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 4 BFC5  gene… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 5 BFC6  Dros… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 NA <NA>
    ## 6 BFC7  txpR… 2024-05-15… 2024-05-15… "F:\… rela… 2098…                 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

    library(devtools)

    ## Loading required package: usethis

    session_info()

    ## ─ Session info ───────────────────────────────────────────────────────────────
    ##  setting  value
    ##  version  R version 4.4.0 (2024-04-24 ucrt)
    ##  os       Windows Server 2022 x64 (build 20348)
    ##  system   x86_64, mingw32
    ##  ui       RTerm
    ##  language (EN)
    ##  collate  C
    ##  ctype    English_United States.utf8
    ##  tz       America/New_York
    ##  date     2024-05-14
    ##  pandoc   2.7.3 @ C:/PROGRA~1/Pandoc/ (via rmarkdown)
    ##
    ## ─ Packages ───────────────────────────────────────────────────────────────────
    ##  ! package              * version     date (UTC) lib source
    ##    abind                  1.4-5       2016-07-21 [2] CRAN (R 4.4.0)
    ##    AnnotationDbi        * 1.66.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    AnnotationFilter       1.28.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    AnnotationHub          3.12.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##  D archive                1.1.8       2024-04-28 [2] CRAN (R 4.4.0)
    ##    Biobase              * 2.64.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    BiocFileCache        * 2.12.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    BiocGenerics         * 0.50.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    BiocIO                 1.14.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    BiocManager            1.30.23     2024-05-04 [2] CRAN (R 4.4.0)
    ##    BiocParallel           1.38.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    BiocVersion            3.19.1      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    biomaRt                2.60.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    Biostrings             2.72.0      2024-05-14 [2] Bioconductor 3.19 (R 4.4.0)
    ##    bit                    4.0.5       2022-11-15 [2] CRAN (R 4.4.0)
    ##    bit64                  4.0.5       2020-08-30 [2] CRAN (R 4.4.0)
    ##    bitops                 1.0-7       2021-04-24 [2] CRAN (R 4.4.0)
    ##    blob                   1.2.4       2023-03-17 [2] CRAN (R 4.4.0)
    ##    bslib                  0.7.0       2024-03-29 [2] CRAN (R 4.4.0)
    ##    cachem                 1.0.8       2023-05-01 [2] CRAN (R 4.4.0)
    ##    cli                    3.6.2       2023-12-11 [2] CRAN (R 4.4.0)
    ##    codetools              0.2-20      2024-03-31 [2] CRAN (R 4.4.0)
    ##    colorspace             2.1-0       2023-01-23 [2] CRAN (R 4.4.0)
    ##    crayon                 1.5.2       2022-09-29 [2] CRAN (R 4.4.0)
    ##    curl                   5.2.1       2024-03-01 [2] CRAN (R 4.4.0)
    ##    DBI                    1.2.2       2024-02-16 [2] CRAN (R 4.4.0)
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# 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.