Recent development of high-throughput sequencing technology has generated large amounts of data at transcriptome level such as RNA-binding protein target sites, RNA modifications and other RNA-related genomic features. These transcriptome attributes can be converted to a set of locations or regions.
Currently, colocalization analysis of two genomic region sets has been widely used to infer potential interactions between corresponding biological attributes. A number of methods have been developed for this. For example, regioneR has been created to statistically estimate association between genomic region sets using a permutation test framework. All its functions are genome and mask-aware. However, there has been no such tools enable researchers to make data analysis and interpretation at transcriptome level. Here RgnTX is invented to fill this blank.
RgnTX extends an assumption-free permutation test framework transcriptome-wide. Different from existing approaches, RgnTX allows the integration of transcriptome annotations so as to model the complex alternative splicing patterns. Importantly, it also supports the testing of transcriptome elements without clear isoform association, which is often the real scenario due to technical limitations.
RgnTX makes a useful tool for transcriptome region set association analysis. In this vignette, with some simulated datasets, we show that permutation tests conducted in the genome and the transcriptome are significantly different and may return distinct results in section 2. The most core functions for conducting permutation tests and examples with real datasets are introduced in section 6.
Suppose we want to know if a region set A has some association with another region set B. We could pick some random region sets, and calculate the number of overlaps between them and B. If the overlap numbers of these random region sets and B are generally smaller than that of A and B, it is more likely to say there exists some association between A and B, otherwise not.
Such permutation test framework mainly involves two steps: the randomization step (picking random region sets) and the evaluation step (evaluating overlapping counts or other test statistic). Importantly, performing it over the transcriptome and the genome can be different in both of these two steps.
Compared with the linear genome space, transcriptome space is heterogeneous due to the existence of multiple isoform transcripts, the splicing junctions and exons used by different frequencies. The isoform specificity of transcriptome elements is often lost in the process of mapping due to technical limitations. To perform isoform-aware permutation for transcriptome element with isoform ambiguity, RgnTX considers only the transcripts that overlap with it when projected to the genome, so as to retrain maximal isoform information. In this example shown by the following figures, feature 3 will be permutated on transcript Tx3 only, while feature 2 will be permutated on all the 3 transcripts.
This is supported by the RgnTX function randomizeFeaturesTx
and the function randomizeFeaturesTxIA
. The former is for features with known isoform belongings. The latter is for features with isoform ambiguity (IA). Besides, RgnTX also provides function randomizeTx
supporting picking random region sets within any specified transcriptome space. Users can choose a suitable strategy to do randomization conveniently within only a few lines of codes. More details about this are introduced in section 5.
Another non-trivial task is to develop evaluation function for a test statistic, such as the number of overlaps, to be tested between two region sets. A key limitation of existing evaluation approaches is that, they were designed primarily for genome-based evaluation. Two transnscriptome elements with shared exons but are located on the independent transcripts are also considered to have overlaps by them. The information of transcripts is totally lost during comparison, which can induce substantial bias when applying to analyze transcriptome region sets.
RgnTX provides several strategies for transcriptome-level evaluation. Function overlapCountsTX
counts the number of overlaps between two transcriptome elements in the transcriptome directly. Function overlapCountsTXIA
calculates a weighted number of overlaps between a feature set (with isoform ambiguity) and a transcriptome region set (without isoform ambiguity). It is also possible to write a custom evaluation function and pass it to the main permutation test function. Details about this part are introduced in section 7.
We perform a series of simulations to show the difference of permutation tests conducted in the transcriptome and in the genome.
In this simulation, we randomly generated 1000 pairs of region sets in the following four spaces, with each region set containing 500 regions of 200nt long on the same 300 genes, and then count the number of overlaps between each pair.
DNA: This space contains randomly picked 300 genes from a hg19 TxDb object.
pre-mRNA: It contains all of the pre-mRNAs that pertain to the previously picked 300 genes.
mRNA: It contains all of the mRNAs that pertain to the previously picked genes.
Exonic DNA: This space involves only the part of genome that is related to the previously picked mRNAs. It is actually a linear space of these mRNAs.
Figure 4 shows the evaluation results. In the first four cases, overlaps are counted based on the genome (or after projection). In the last case, overlaps are counted in the transcriptome directly. The former four cases are evaluated by regioneR function numOverlaps
, while the last is evaluated by RgnTX function overlapCountsTx
.
As the results show, permutations over different genome and transcriptome space return distinct results. A small number of overlaps are observed in the transcriptome (the last case), which is not surprising due to the existence of multiple isoform transcripts. Interestingly, the results from exonic DNA and mRNA (projected to genome) are also different, which is due to exons used at different frequencies by isoform transcripts. These results suggest that the permutation of heterogeneous transcriptome elements can be substantially more complex than genome-based elements. Genome-based methods are not appropriate to analyze transcriptome elements. That’s why we develop RgnTX to do this.
To install this package via devtools, please use the following codes.
if (!requireNamespace("devtools", quietly = TRUE))
install.packages("devtools")
devtools::install_github("yue-wang-biomath/RgnTX", build_vignettes = TRUE)
To install this package via BiocManager, please use the following codes.
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("RgnTX")
RgnTX is based on the GenomicRanges and GenomicFeatures structure. In this section, we introduce the format of region sets and other associated metadata used in RgnTX.
A TxDb object stores transcripts, exons, CDS and other types of genomic features. A TxDb object is usually required by RgnTX functions. Currently, all the examples in this vignette are based on the TxDb.Hsapiens.UCSC.hg19.knownGene
. Users can assign other TxDb objects via related function arguments.
A TxDb object provides default ids and names for transcripts. According to GenomicFeatures, a transcript is guaranteed to be related to a unique transcript id. But a transcript’s name is not guaranteed to be unique or even defined. In this vignette, all the examples use transcript ids instead of names to indicate specific transcripts.
Please pay attention not to confuse the transcript ids with other types of ids, such as ids for CDS, exons and other genomic features provided by a TxDb object, or external ids for genes like Entrez Gene and Ensembl ids. Functions in RgnTX will not work with wrong types of ids.
The feature/region sets in RgnTX should be either GRanges
or GRangesList
.
For a GRangesList
feature set, every list element that contains a set of ranges is a feature. For a GRanges
feature set without special instructions, a single range is a feature. For a GRanges
feature set containing a metadata column named “group”, the set of ranges having the same group number is a feature.
The transcript ids (if available) should be provided as follows. For GRangesList
objects, the name of each list element (feature) should be the id of the transcript that this feature is located on. For GRanges
objects, transcript ids should be contained in a metadata column named “transcriptsHits”.
Here is an example of GRangesList
feature set following the format required by RgnTX. Its names should be transcript ids.
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
set.seed(12345677)
example.list <- randomizeTx(txdb, random_num = 10, random_length = 200)
example.list
## GRangesList object of length 10:
## $`9974`
## GRanges object with 1 range and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr2 202725672-202725871 +
## -------
## seqinfo: 8 sequences from an unspecified genome; no seqlengths
##
## $`52204`
## GRanges object with 2 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr14 104143758-104143860 +
## [2] chr14 104145721-104145817 +
## -------
## seqinfo: 8 sequences from an unspecified genome; no seqlengths
##
## $`73524`
## GRanges object with 2 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr22 18893876-18893997 +
## [2] chr22 18894078-18894155 +
## -------
## seqinfo: 8 sequences from an unspecified genome; no seqlengths
##
## ...
## <7 more elements>
The following GRanges
object represents the same region set as the above one. Ranges having the same group number represent a feature. The transcriptsHits
column contains corresponding transcript ids. Both formats are accepted by RgnTX functions and can be transformed to each other via function GRangesList2GRanges
and function GRanges2GRangesList
.
example.gr <- GRangesList2GRanges(example.list)
example.gr
## GRanges object with 17 ranges and 2 metadata columns:
## seqnames ranges strand | transcriptsHits group
## <Rle> <IRanges> <Rle> | <character> <integer>
## [1] chr2 202725672-202725871 + | 9974 1
## [2] chr14 104143758-104143860 + | 52204 2
## [3] chr14 104145721-104145817 + | 52204 2
## [4] chr22 18893876-18893997 + | 73524 3
## [5] chr22 18894078-18894155 + | 73524 3
## ... ... ... ... . ... ...
## [13] chr17 73917325-73917345 - | 64316 8
## [14] chr17 73916355-73916510 - | 64316 8
## [15] chr17 73916166-73916188 - | 64316 8
## [16] chr12 122989759-122989958 - | 49210 9
## [17] chr7 121960010-121960209 - | 31300 10
## -------
## seqinfo: 8 sequences from an unspecified genome; no seqlengths
example.list <- GRanges2GRangesList(example.gr)
In RgnTX, there are mainly two kinds of randomization functions. One can pick random regions over a specified space (see section 5.1). One can also randomize specified features into transcriptome, i.e., input a set of features and get a randomized result of it (see section 5.2).
In this strategy, users need to specify a scope that random region sets will be picked from.
In this function, users can pass transcript ids via an argument to make random regions to be picked from the transcripts having these ids.
Step 1: specify a randomization space
One needs to determine three arguments: a TxDb data, a set of transcript ids and the type of randomized regions the function will return. As an example, we specify a randomization space formed by five mRNAs.
txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene
type <- "mature"
trans.ids <- c("170", "782", "974", "1364", "1387")
We feed them into the randomizeTX
function.
Step 2: randomizeTx function
We use this function to pick 10 random regions, and each region is 100 nt long. Each region is picked from a random transcript among the input transcripts.
set.seed(12345677)
randomResults <- randomizeTx(txdb, trans_ids = trans.ids, random_num = 10,
random_length = 100)
txdb: a TxDb obejct.
trans_ids (default: “all”): the ids of transcripts, which should be a character object. If this argument takes the default value all
, random regions will be picked from the whole transcriptome.
random_num: the number of regions to be picked.
random_length: the length of each region to be picked.
type (default: “mature”): this argument receives options mature
, full
, fiveUTR
, CDS
or threeUTR
, with which users can get corresponding types of random regions. Default is “mature”, with which the function picks regions over mRNAs.
N (default: 1): randomization times. The number of sets of region sets the function will return.
randomResults
## GRangesList object of length 10:
## $`1364`
## GRanges object with 1 range and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr1 54433399-54433498 +
## -------
## seqinfo: 1 sequence from an unspecified genome; no seqlengths
##
## $`170`
## GRanges object with 2 ranges and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr1 3599695-3599744 +
## [2] chr1 3624113-3624162 +
## -------
## seqinfo: 1 sequence from an unspecified genome; no seqlengths
##
## $`170`
## GRanges object with 1 range and 0 metadata columns:
## seqnames ranges strand
## <Rle> <IRanges> <Rle>
## [1] chr1 3638649-3638748 +
## -------
## seqinfo: 1 sequence from an unspecified genome; no seqlengths
##
## ...
## <7 more elements>
width(randomResults)
## IntegerList of length 10
## [["1364"]] 100
## [["170"]] 50 50
## [["170"]] 100
## [["974"]] 6 94
## [["974"]] 100
## [["974"]] 100
## [["1387"]] 100
## [["1387"]] 100
## [["1364"]] 63 37
## [["170"]] 100
The function returns a GRangesList
object. Every list element has a total length of 100 nt and represents a region on mRNA but possibly separated by intron parts. The name of each element is its relative transcript id.
This is another randomization function. The difference of this function and randomizeTx
is that it does not take an argument about transcript ids, instead, it takes a GRangesList
region set and picks only one random region within the space indicated by every GRangesList
element. So the number of regions in a set this function returns is equal to the number of elements in the input GRangesList
object.
Step 1: specify a randomization space
We pick a set of five mRNAs to be the scope of randomization.
trans.ids <- c("170", "782", "974", "1364", &qu