---
title: "Importing transcript abundance with tximport"
author: "Michael I. Love, Charlotte Soneson, Mark D. Robinson"
date: "`r Sys.Date()`"
package: "`r packageVersion('tximport')`"
output:
  rmarkdown::html_document:
    highlight: pygments
    toc: true
    toc_float: true
    fig_width: 5
bibliography: library.bib
vignette: >
  %\VignetteIndexEntry{Importing transcript abundance datasets with tximport}
  %\VignetteEngine{knitr::rmarkdown}
---

## Introduction

Import and summarize transcript-level abundance estimates for
transcript- and gene-level analysis with Bioconductor packages, such as *edgeR*,
*DESeq2*, and *limma-voom*.
The motivation and methods for the functions
provided by the *tximport* package are described in the following
article [@Soneson2015]:

> Charlotte Soneson, Michael I. Love, Mark D. Robinson (2015):
> Differential analyses for RNA-seq: transcript-level estimates
> improve gene-level inferences. *F1000Research*
> http://dx.doi.org/10.12688/f1000research.7563.1

In particular, the *tximport* pipeline offers the following benefits:
(i) this approach corrects for potential changes in gene length across samples 
(e.g. from differential isoform usage) [@Trapnell2013Differential],
(ii) some of the upstream quantification methods (*Salmon*, *Sailfish*, *kallisto*) 
are substantially faster and require less memory
and disk usage compared to alignment-based methods that require
creation and storage of BAM files, and
(iii) it is possible to avoid discarding those fragments that can
align to multiple genes with homologous sequence, thus increasing
sensitivity [@Robert2015Errors].

**Note:** another Bioconductor package, 
[tximeta](https://bioconductor.org/packages/tximeta) [@Love2020],
extends *tximport*, offering the same functionality, plus the
additional benefit of automatic addition of annotation metadata for
commonly used transcriptomes (GENCODE, Ensembl, RefSeq for human and
mouse). See the [tximeta](https://bioconductor.org/packages/tximeta)
package vignette for more details. Whereas `tximport` outputs a simple
list of matrices, `tximeta` will output a *SummarizedExperiment*
object with appropriate *GRanges* added if the transcriptome is from
one of the sources above for human and mouse. *tximeta* also offers
easy conversion to data objects used by *edgeR* and *limma* with the
`makeDGEList` function.

```{r, echo=FALSE}
library(knitr)
opts_chunk$set(tidy=TRUE,message=FALSE)
```

## Import transcript-level estimates

We begin by locating some prepared files that contain
transcript abundance estimates for six samples, from the
*tximportData* package. The *tximport* pipeline will be nearly
identical for various quantification tools, usually only
requiring one change the `type` argument.
We begin with quantification files generated by the *Salmon*
software, and later show the use of *tximport*
with any of: 

* *Salmon* [@Patro2017Salmon]
* *Alevin* [@Srivastava2019]
* *Sailfish* [@Patro2014Sailfish]
* *kallisto* [@Bray2016Near]
* *RSEM* [@Li2011RSEM]
* *StringTie* [@Pertea2015]

First, we locate the directory containing the files. (Here we use
`system.file` to locate the package directory, but for a typical use,
we would just provide a path, e.g. `"/path/to/dir"`.)

```{r}
library(tximportData)
dir <- system.file("extdata", package="tximportData")
list.files(dir)
```

Next, we create a named vector pointing to the quantification
files. We will create a vector of filenames first by reading in a
table that contains the sample IDs, and then combining this with `dir`
and `"quant.sf.gz"`. (We gzipped the quantification files to make the
data package smaller, this is not a problem for R functions that we
use to import the files.)

```{r}
samples <- read.table(file.path(dir,"samples.txt"), header=TRUE)
samples
files <- file.path(dir, "salmon", samples$run, "quant.sf.gz")
names(files) <- paste0("sample",1:6)
all(file.exists(files))
```

Transcripts need to be associated with gene IDs for gene-level
summarization. If that information is present in the files, we can
skip this step. For Salmon, Sailfish, and kallisto the files only
provide the transcript ID.  We first make a data.frame called
`tx2gene` with two columns: 1) transcript ID and 2) gene ID. The column names do not
matter but this column order must be used.  The transcript ID must be
the same one used in the abundance files. 

Creating this `tx2gene` data.frame can be accomplished from a *TxDb* object
and the `select` function from the *AnnotationDbi* package. The
following code could be used to construct such a table:

```{r}
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
k <- keys(txdb, keytype="TXNAME")
tx2gene <- select(txdb, k, "GENEID", "TXNAME")
```

Note: if you are using an *Ensembl* transcriptome, the easiest way to
create the `tx2gene` data.frame is to use the
[ensembldb](http://bioconductor.org/packages/ensembldb) packages.
The annotation packages can be found by version number, and use
the pattern `EnsDb.Hsapiens.vXX`. The `transcripts` function can
be used with `return.type="DataFrame"`, in order to obtain
something like the `df` object constructed in the code chunk above.
See the *ensembldb* package vignette for more details.

In this case, we've used the Gencode v27 CHR transcripts to build our
index, and we used `makeTxDbFromGFF` and code similar to the chunk
above to build the `tx2gene` table. We then read in a pre-constructed
`tx2gene` table:

```{r}
library(readr)
tx2gene <- read_csv(file.path(dir, "tx2gene.gencode.v27.csv"))
head(tx2gene)
```

The *tximport* package has a single function for importing
transcript-level estimates.  The `type` argument is used to specify
what software was used for estimation.  A simple list with
matrices, `"abundance"`, `"counts"`, and `"length"`, is returned, where the
transcript level information is summarized to the
gene-level. Typically, abundance is provided by the quantification
tools as TPM (transcripts-per-million), while the counts are estimated
counts (possibly fractional), and the `"length"` matrix contains the
effective gene lengths.
The `"length"` matrix can be used to generate an offset matrix for
downstream gene-level differential analysis of count matrices, as
shown below.

**Note**: While *tximport* works without any dependencies, it is
significantly faster to read in files using the *readr* package.  If
*tximport* detects that *readr* is installed, then it will use the
`readr::read_tsv` function by default. A change from version 1.2 to
1.4 is that the reader is not specified by the user anymore, but
chosen automatically based on the availability of the *readr*
package. Advanced users can still customize the import of files using
the `importer` argument.

```{r}
library(tximport)
txi <- tximport(files, type="salmon", tx2gene=tx2gene)
names(txi)
head(txi$counts)
```

We could alternatively generate counts from abundances, using the
argument `countsFromAbundance`, scaled to library size, `"scaledTPM"`,
or additionally scaled using the average transcript length, averaged
over samples and to library size, `"lengthScaledTPM"`.  Using either
of these approaches, the counts are not correlated with length, and so
the length matrix should not be provided as an offset for
downstream analysis packages. As of *tximport* version 1.10, we have
added a new `countsFromAbundance` option `"dtuScaledTPM"`. This
scaling option is designed for use with `txOut=TRUE` for differential
transcript usage analyses. See `?tximport` for details on the various
`countsFromAbundance` options.

We can avoid gene-level summarization by setting `txOut=TRUE`, giving
the original transcript level estimates as a list of matrices.

```{r}
txi.tx <- tximport(files, type="salmon", txOut=TRUE)
```

These matrices can then be summarized afterwards using the function
`summarizeToGene`. This then gives the identical list of matrices as using
`txOut=FALSE` (default) in the first `tximport` call.

```{r}
txi.sum <- summarizeToGene(txi.tx, tx2gene)
all.equal(txi$counts, txi.sum$counts)
```

## Salmon

Salmon or Sailfish `quant.sf` files can be imported by setting type to
`"salmon"` or `"sailfish"`.

```{r}
files <- file.path(dir,"salmon", samples$run, "quant.sf.gz")
names(files) <- paste0("sample",1:6)
txi.salmon <- tximport(files, type="salmon", tx2gene=tx2gene)
head(txi.salmon$counts)
```

We quantified with Sailfish against a different transcriptome, so we
need to read in a different `tx2gene` for this next code chunk.

```{r}
tx2knownGene <- read_csv(file.path(dir, "tx2gene.csv"))
files <- file.path(dir,"sailfish", samples$run, "quant.sf")
names(files) <- paste0("sample",1:6)
txi.sailfish <- tximport(files, type="sailfish", tx2gene=tx2knownGene)
head(txi.sailfish$counts)
```

*Note*: for previous version of Salmon or Sailfish, in which the
`quant.sf` files start with comment lines, it is recommended to
specify the `importer` argument as a function which reads in the 
lines beginning with the header. For example, using the following
code chunk (un-evaluated): 
 
```{r eval=FALSE}
txi <- tximport("quant.sf", type="none", txOut=TRUE,
                txIdCol="Name", abundanceCol="TPM",
                countsCol="NumReads", lengthCol="Length",
                importer=function(x) read_tsv(x, skip=8))
```

## Salmon with inferential replicates

If inferential replicates (Gibbs or bootstrap samples) are present in
expected locations relative to the `quant.sf` file, *tximport* will 
import these as well. Here we demonstrate using Salmon, run with only
5 Gibbs replicates (usually more Gibbs samples, such as 20 or 30,
would be useful for capturing inferential uncertainty). 

```{r}
files <- file.path(dir,"salmon_gibbs", samples$run, "quant.sf.gz")
names(files) <- paste0("sample",1:6)
txi.inf.rep <- tximport(files, type="salmon", txOut=TRUE)
names(txi.inf.rep)
names(txi.inf.rep$infReps)
dim(txi.inf.rep$infReps$sample1)
```

The *tximport* arguments `varReduce` and `dropInfReps` can be used to
summarize the inferential replicates into a single variance per
transcript/gene and per sample, or to not import inferential
replicates, respectively. 

## kallisto

kallisto `abundance.h5` files can be imported by setting type to
`"kallisto"`. Note that this requires that you have the Bioconductor package 
[rhdf5](http://bioconductor.org/packages/rhdf5) installed.
(Here we only demonstrate reading in transcript-level information.)

```{r}
files <- file.path(dir, "kallisto_boot", samples$run, "abundance.h5")
names(files) <- paste0("sample",1:6)
txi.kallisto <- tximport(files, type="kallisto", txOut=TRUE)
head(txi.kallisto$counts)
```

## kallisto with inferential replicates

Because the `kallisto_boot` directory also has inferential replicate
information, it was imported as well.

```{r}
names(txi.kallisto)
names(txi.kallisto$infReps)
dim(txi.kallisto$infReps$sample1)
```

## kallisto with TSV files

kallisto `abundance.tsv` files can be imported as well, but this is
typically slower than the approach above. Note that we add an
additional argument in this code chunk, `ignoreAfterBar=TRUE`. This is
because the Gencode transcripts have names like
"ENST00000456328.2|ENSG00000223972.5|...", though our `tx2gene` table
only includes the first "ENST" identifier. We therefore want to split
the incoming quantification matrix rownames at the first bar "|", and
only use this as an identifier. We didn't use this option earlier with
Salmon, because we used the argument `--gencode` when running Salmon,
which itself does the splitting upstream of `tximport`. Note that 
`ignoreTxVersion` and `ignoreAfterBar` are only to facilitating the
summarization to gene level.

```{r}
files <- file.path(dir, "kallisto", samples$run, "abundance.tsv.gz")
names(files) <- paste0("sample",1:6)
txi.kallisto.tsv <- tximport(files, type="kallisto", tx2gene=tx2gene,
                             ignoreAfterBar=TRUE)
head(txi.kallisto.tsv$counts)
```

## RSEM

RSEM `sample.genes.results` files can be imported by setting
type to `"rsem"`, and `txIn` and `txOut` to `FALSE`.

```{r}
files <- file.path(dir,"rsem", samples$run, paste0(samples$run, ".genes.results.gz"))
names(files) <- paste0("sample",1:6)
txi.rsem <- tximport(files, type="rsem", txIn=FALSE, txOut=FALSE)
head(txi.rsem$counts)
```

RSEM `sample.isoforms.results` files can be imported by setting
type to `"rsem"`, and `txIn` and `txOut` to `TRUE`.

```{r}
files <- file.path(dir,"rsem", samples$run, paste0(samples$run, ".isoforms.results.gz"))
names(files) <- paste0("sample",1:6)
txi.rsem <- tximport(files, type="rsem", txIn=TRUE, txOut=TRUE)
head(txi.rsem$counts)
```

## StringTie

StringTie `t_data.ctab` files giving the coverage and abundances for
transcripts can be imported by setting type to `stringtie`. These
files can be generated with the following command line call:

```
stringtie -eB -G transcripts.gff <source_file.bam> 
```

*tximport* will compute counts from the coverage information, by
reversing the formula that StringTie uses to calculate coverage (see
`?tximport`). The read length is used in this formula, and so if
you've set a different read length when using StringTie, you can
provide this information with the `readLength` argument. The `tx2gene`
table should connect transcripts to genes, and can be pulled out of
one of the `t_data.ctab` files. The tximport call would look like the
following (here not evaluated): 

```{r, eval=FALSE}
tmp <- read_tsv(files[1])
tx2gene <- tmp[,c("t_name","gene_name")]
txi <- tximport(files, type="stringtie", tx2gene=tx2gene)
```

## Alevin

scRNA-seq data quantified with *Alevin* can be easily imported using
*tximport*. The following unevaluated example shows import of the
quants matrix (for a live example, see the unit test file
`test_alevin.R`). A single file should be specified which will import
a gene-by-cell matrix of data.

```{r, eval=FALSE}
files <- "path/to/alevin/quants_mat.gz"
txi <- tximport(files, type="alevin")
```

## Downstream DGE in Bioconductor

**Note**: there are two suggested ways of importing estimates for use
with differential gene expression (DGE) methods. The first method,
which we show below for *edgeR* and for *DESeq2*, is to use the
gene-level estimated counts from the quantification tools, and additionally to
use the transcript-level abundance estimates to calculate a gene-level offset
that corrects for changes to the average transcript length across
samples. The code examples below accomplish these steps for you,
keeping track of appropriate matrices and calculating these
offsets. For *edgeR* you need to assign a matrix to `y$offset`, but
the function *DESeqDataSetFromTximport* takes care of creation of the
offset for you. Let's call this method "*original counts and offset*".

The second method is to use the `tximport` argument
`countsFromAbundance="lengthScaledTPM"` or `"scaledTPM"`, and then to
use the gene-level count matrix `txi$counts` directly as you would a regular
count matrix with these software. Let's call this method 
"*bias corrected counts without an offset*"

**Note:** Do not manually pass the original gene-level counts
to downstream methods *without an offset*. The only case where this
would make sense is if there is no length bias to the counts, as 
happens in 3' tagged RNA-seq data (see section below).
The original gene-level counts are in `txi$counts` when `tximport` was run with
`countsFromAbundance="no"`. This is simply passing the summed
estimated transcript counts, and does not correct for potential differential
isoform usage (the offset), which is the point
of the *tximport* methods [@Soneson2015] for gene-level analysis. Passing
uncorrected gene-level counts without an offset is not recommended by the
*tximport* package authors. The two methods we provide here are: 
"*original counts and offset*" or "*bias corrected counts without an
offset*". Passing `txi` to `DESeqDataSetFromTximport` as
outlined below is correct: the function creates the appropriate offset
for you to perform gene-level differential expression.

## 3' tagged RNA-seq

If you have 3' tagged RNA-seq data, then correcting the
counts for gene length will induce a bias in your analysis, because
the counts do not have length bias. Instead of using the default
full-transcript-length pipeline, we recommend to use the
original counts, e.g. `txi$counts` as a counts matrix, e.g. providing
to *DESeqDataSetFromMatrix* or to the *edgeR* or *limma* functions
without calculating an offset and without using *countsFromAbundance*.

## edgeR

An example of creating a `DGEList` for use with *edgeR*
[@Robinson2010] follows. Note that the alternate package, *tximeta*,
described above has a convenience function `makeDGEList` for creating
a data object for use with *edgeR* and *limma*.

```{r, results="hide", messages=FALSE}
library(edgeR)
```

```{r}
cts <- txi$counts
normMat <- txi$length

# Obtaining per-observation scaling factors for length,
# adjusted to avoid changing the magnitude of the counts.
normMat <- normMat / exp(rowMeans(log(normMat)))
normCts <- cts / normMat

# Computing effective library sizes from scaled counts,
# to account for composition biases between samples.
library(edgeR)
eff.lib <- calcNormFactors(normCts) * colSums(normCts)

# Combining effective library sizes with the length factors,
# and calculating offsets for a log-link GLM.
normMat <- sweep(normMat, 2, eff.lib, "*")
normMat <- log(normMat)

# Creating a DGEList object for use in edgeR.
y <- DGEList(cts)
y <- scaleOffset(y, normMat)
# filtering
keep <- filterByExpr(y)
y <- y[keep,]
# y is now ready for estimate dispersion functions
# see edgeR User's Guide
```

For creating a matrix of CPMs within *edgeR*, the following code chunk can be used:

```{r}
cpms <- edgeR::cpm(y, offset=y$offset, log=FALSE)
```

## DESeq2

An example of creating a `DESeqDataSet` for use with *DESeq2* [@Love2014]:

```{r, results="hide", messages=FALSE}
library(DESeq2)
```

The user should make sure the rownames of `sampleTable` align with the
colnames of `txi$counts`, if there are colnames. The best practice is
to read `sampleTable` from a CSV file, and to construct `files` from a
column of `sampleTable`, as was shown in the *tximport* examples above.

```{r}
sampleTable <- data.frame(condition=factor(rep(c("A","B"),each=3)))
rownames(sampleTable) <- colnames(txi$counts)
```

```{r}
dds <- DESeqDataSetFromTximport(txi, sampleTable, ~ condition)
# dds is now ready for DESeq()
# see DESeq2 vignette
```

## limma-voom

An example of creating a data object for use with *limma-voom* [@Law2014]. 
Because limma-voom does not use the offset matrix stored in
`y$offset`, we recommend using the scaled counts generated from
abundances, either `"scaledTPM"` or `"lengthScaledTPM"`:

```{r}
files <- file.path(dir,"salmon", samples$run, "quant.sf.gz")
names(files) <- paste0("sample",1:6)
txi <- tximport(files, type="salmon",
                tx2gene=tx2gene,
                countsFromAbundance="lengthScaledTPM")
library(limma)
y <- DGEList(txi$counts)
# filtering
keep <- filterByExpr(y)
y <- y[keep,]
y <- calcNormFactors(y)
design <- model.matrix(~ condition, data=sampleTable)
v <- voom(y, design)
# v is now ready for lmFit()
# see limma User's Guide
```

## Acknowledgments

The development of *tximport* has benefited from contributions and
suggestions from:

[Rob Patro](https://twitter.com/nomad421) (inferential replicates import),
[Andrew Parker Morgan](https://github.com/andrewparkermorgan) (RHDF5 support),
[Ryan C. Thompson](https://github.com/DarwinAwardWinner) (RHDF5 support),
[Matt Shirley](https://twitter.com/mdshw5) (ignoreTxVersion),
[Avi Srivastava](https://twitter.com/k3yavi) (`alevin` import),
Scott Van Buren (infReps testing),
[Stephen Turner](https://twitter.com/genetics_blog),
[Richard Smith-Unna](https://twitter.com/blahah404),
[Rory Kirchner](https://twitter.com/RoryKirchner),
[Martin Morgan](https://twitter.com/mt_morgan),
Jenny Drnevich,
[Patrick Kimes](https://twitter.com/pkkimes),
[Leon Fodoulian](https://twitter.com/LFodoulian),
[Koen Van den Berge](https://twitter.com/koenvdberge_Be),
[Aaron Lun](https://github.com/LTLA),
[Alexander Toenges](https://github.com/ATpoint)

## Session info

```{r}
sessionInfo()
```
 
## References

