We released a flexible plotting tool, sashimi_plot, for visualizing RNA-Seq data and MISO output. Check it out!
MISO (Mixture-of-Isoforms) is a probabilistic framework that quantitates the expression level of alternatively spliced genes from RNA-Seq data, and identifies differentially regulated isoforms or exons across samples. By modeling the generative process by which reads are produced from isoforms in RNA-Seq, the MISO model uses Bayesian inference to compute the probability that a read originated from a particular isoform.
The MISO framework is described in Katz et. al., Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nature Methods (2010).
MISO treats the expression level of a set of isoforms as a random variable and estimates a distribution over the values of this variable. The estimation algorithm is based on sampling, and falls in the family of techniques known as Markov Chain Monte Carlo (“MCMC”). For details of the inference procedure, see Katz et. al. (2010).
Mailing list where users can ask technical questions is available at miso-users (http://mailman.mit.edu/mailman/listinfo/miso-users).
Thu, Dec 20: Posted new mouse genome annotation for alternative cleavage and polyadenylation events (TandemUTR) and alternative last exon events (ALE) from Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing (Nature Methods, Dec. 2012), see Expanded alternative cleavage and polyadenylation annotation for mouse. Thanks to Wencheng Li and Bin Tian for creating this annotation!
Tue, Dec 4: Posted mappings from alternative events to genes (see Mapping of alternative events to genes) and GFF annotations for isoform-centric inference (see Human/mouse gene models for isoform-centric analyses).
Thu, Sept 27: Released misopy-0.4.6. This release fixes a packaging error with sashimi_plot (test case files were omitted.) No changes were made to MISO. Thanks to Schragi Schwartz and Rahul Satija.
Tue, Sept 4: Released misopy-0.4.5. Fixed error in parsing of settings file.
Thur, Jul 26: Released misopy-0.4.4. Turned off autoconvergence features of sampler that caused delayed run times on certain events. (Also introduced a parameter num_chains in MISO settings file to control the number of independent MCMC chains used by the sampler.)
Fri, Jun 1: Released misopy-0.4.3. Included one main bug fix to an instability issue related to Bayes factors which affected low coverage events and better error handling for sashimi_plot. The Bayes factor bug resulted in some very low coverage events having highly variable Bayes factor values across MISO runs. This issue does not affect the Ψ values or their confidence intervals, only the Bayes factor values. Events that meet most standard coverage filters applied to read counts will not be affected. Thanks to Ersen Kavak, Essi Laajala and Schragi Schwartz.
Fri, May 4: Release of misopy-0.4.2. Included some bug fixes, notably:
Thanks to Rob Bradley, Eric Suh, Brad Friedman and others for their comments.
Wed, Feb 1: Release of misopy-0.4.1. This is mainly a feature release. Updates include gene information being outputted in summary files and sample comparison files, dynamic scaling for sashimi_plot (described in its documentation page), options for labeling summary/comparison files, and other minor features. Bugfixes include: correct inclusion of test files with pypi releases, correction of edge cases that caused compare-samples to fail in case a multi-isoform event was improperly sampled.
Fri, Jan 13: Release misopy-0.4. Includes mainly fix to serious bug that caused MISO to drop junction reads for events containing alignments with insertions (e.g. CIGAR strings containing I). Users of Tophat and other read mappers that allow insertions in alignments please take notice.
Sun, Jan 8: Release misopy-0.3. Includes mainly sashimi_plot features/bugfixes.
Tue, Jan 3: Released misopy-0.2. Includes bugfixes to test cases and new sashimi_plot features.
Mon, Dec 26: We’ve done some major reorganization of MISO and sashimi_plot to make them official Python packages. MISO can now be installed like as a standard Python package from the Python Package Index (see our pypi page.) The manual has been changed to reflect the new and improved installation procedure, which makes it easier to invoke MISO from the command line, requiring no modifications to your environment path variables.
The new reorganized version of MISO cannot be used with older indexed/pickled files, and requires GFF files to be re-indexed by calling index_gff.py from the new package. Indexed/pickled files created with older versions of MISO will not work with the new version. We apologize for the inconvenience. Future updates to MISO will not need this re-indexing step, but our refactoring of the code results in this being required for this upgrade.
Fri, Dec 16: Added support for cluster submission using SGE – thanks to Michael Lovci for implementing this feature. Fixed edge case that caused filter_events to fail, thanks to Kuan-Ting Lin for pointing this out.
Wed, Dec 14: Several new updates, including:
Sun, Dec 11: Removed platform-specific pickled files from annotation zip files.
Sat, Dec 3: sashimi_plot, a tool for visualizing RNA-Seq reads along exons/junctions as well as MISO output is released!
Fri, Dec 2: Underscores (_) are now allowed in GFF annotations.
Fri, Nov 11 (11/11/11): We posted a link to MISO annotations of alternative events for the Drosophila melanogaster genome, courtesy of Brent Graveley and the modENCODE project. Thanks to Brent for compiling and sharing these helpful annotations! We encourage those who work on Drosophila transcriptome datasets to try these and contribute more annotations of novel alternative RNA processing events.
Thu, Oct 27: Released fastmiso, a version of MISO written in the C programming language which is 60-100x faster than the Python only version. The MISO version contains a Python interface that is identical to the original Python-onlyMISO version, and thus will require no modification on the part of users aside from compiling the C code. An interface to MISO in the R language is also available (currently undocumented.)
Tue, Oct 18: The White House adopts MISO. From the article: “Miso would be so upset if I didn’t tell you what it was,” said Host Chuck Todd employing one of his many puns.
Fri, Sept 9: Human alternative event annotations (in GFF format) for hg19 are posted. Thanks to Brent Graveley for generating these.
The primary version of MISO is called fastmiso – which is written in a combination of Python and the C programming language. This version is approximately 60-100x faster than the older Python-only version of MISO, which is no longer supported. To install fastmiso, see section Installing the fastmiso version of MISO. (To download the Python only version of MISO, see Installing Python-only MISO version.) We recommend using fastmiso.
To install MISO, either download one of the stable releases (see Releases) or install the latest version from our GitHub repository. See below for detailed installation instructions.
MISO is available as a Python package, listed as misopy in pypi (Python Package Index).
MISO requires a small number of Python modules and commonly used software like samtools for accessing SAM/BAM files. The requirements are:
Required Python modules:
Other required software:
We strongly recommend that you install MISO using a Python package manager (see Installing the fastmiso version of MISO) so that the required Python modules are automatically installed and managed for you.
To quickly start using MISO, follow these steps (each of which is described in detail in the rest of the manual):
Choose your GFF annotation set (see Alternative event annotations for our own annotations, available for human, mouse and fruit fly genomes.)
Index the annotation using index_gff.py:
python index_gff.py --index SE.gff3 indexed_SE_events/
where SE.gff3 is a GFF file containing descriptions of isoforms/alternative splicing events to be quantitated (e.g. skipped exons).
Use run_events_analysis.py to get isoform expression estimates, optionally using a cluster to run jobs in parallel, e.g.:python run_events_analysis.py --compute-genes-psi indexed_SE_events/ my_sample1.bam --output-dir my_output1/ --read-len 36 --use-cluster python run_events_analysis.py --compute-genes-psi indexed_SE_events/ my_sample2.bam --output-dir my_output2/ --read-len 36 --use-clusterwhere indexed_SE_events is a directory containing the indexed skipped exon events.
Summarize MISO inferences using run_miso.py --summarize-samples:python run_miso.py --summarize-samples my_output1/ summaries/ python run_miso.py --summarize-samples my_output2/ summaries/
Make pairwise comparisons between samples to detect differentially expressed isoforms/events with run_miso.py --compare-samples:
python run_miso.py --compare-samples my_output1/ my_output2/ comparison_output/
Parse and filter significant events with sufficient coverage.
The fastmiso version of MISO is written in a combination of C and Python. The underlying statistical inference engine is written in C and can be compiled on Mac OS X or Unix-like platforms using GCC or other standard compilers. The interface to this code is written in Python and is identical in its commands and input and output formats to the original Python-only version of MISO. (The Python-only version of MISO is now deprecated.)
There are two options for installing MISO, using either a stable release or installing the latest version from GitHub.
It is highly recommended to install Python packages using a package manager such as the distribute easy_install utility (see Python packaging tools tutorial for straightforward instructions on getting started.) Package managers will automatically download and install all necessary requirements for MISO and save headaches later on. To install a stable release package of MISO using a Python package manager like distribute’s easy_install, simply type:
This will install MISO globally, fetching and installing all of its required Python modules. For local installation, use:
easy_install --user -U misopy
If easy_install is unavailable on your system, you can simply download a stable release package (available from Releases) and install it like ordinary Python modules. Unzip the package (e.g. for version “0.x”):
tar -xzvf misopy-0.x.tar.gz
This will create a setup.py file in the directory where the package was unzipped. Compile the code and install MISO packages as follows:
python setup.py install
This command will install MISO globally on the system. To install it in a particular location, such as your home directory, use the following call to setup.py instead:
python setup.py install --prefix=~/
This will result in a local installation of the MISO Python package. To test the installation, load Python and import the misopy and pysplicing packages in the interpreter:
>> import misopy >> import pysplicing
If you get no errors, the installation completed successfully. If you choose to install packages using --prefix= instead of a package manager, please consider that the Python packages you install have to be visible from your PYTHONPATH (see Python packaging tools tutorial for information.)
MISO needs to samtools to be installed and available on your path.
To install the latest version from the GitHub repository, there are two options: (a) using git to get the latest source from our GitHub repository, or (b) by downloading a snapshot of the current repository, without going through git. If you have git installed and configured with your GitHub account, you can clone our repository as follows:
git clone git://github.com/yarden/MISO.git
This will create a directory called MISO, containing the repository. Alternatively, if you do not want to go through git, you can download a zip file containing the latest MISO GitHub release (following this link: https://github.com/yarden/MISO/zipball/fastmiso). This zip file has to be unzipped (e.g. using unzip fastmiso on Unix systems) and contains the latest release of the MISO repository.
Next, compile the code and install MISO using the Python package manager (such as easy_install) as described above, or using setup.py. With the package manager, use:
easy_install --user -U .
If a package manager is not available, resort to setup.py:
python setup.py install
The --prefix= option can be used as usual to specify an alternative installation path. For example, to install MISO in your local directory /home/user/pylibs/, you can use:
python setup.py install --prefix=/home/user/pylibs/
This will create a directory called lib inside that directory that contains the site-packages directory, which is the directory Python expects to find packages in. The exact path of the directory depends on the Python version used when calling setup.py. If you used Python 2.7, your directory might be /home/user/pylibs/lib/python2.7/site-packages/. To ensure that this directory is available to Python, you can add it to your PYTHONPATH environment variable, as follows (for bash-shell like systems):
This will place the local site-packages ahead of the current setting of PYTHONPATH, resulting in Python loading the version of the package installed there (if it is available) over versions existing in the system’s global site-packages directory.
Executable scripts that are part of MISO (like run_events_analysis.py) get placed in a local bin directory (e.g. ~/bin/) directory. If you’d like these to get used from the shell in place of a globally installed version, make sure that ~/bin is first in your PATH variable.
We strongly recommend that you manage Python packages using a package manager rather than manually installing packages and modifying your PYTHONPATH, which is error prone and time consuming.
To test if the required modules are available, use the module_availability.py script as follows:
$ python module_availability.py Checking for availability of: numpy Checking for availability of: scipy ... All modules are available!
It is crucial that the samtools command is available from the default path environment, or else MISO will not be able to parse SAM/BAM files. On a Unix or a Mac OS X system, you should be able to type samtools at the prompt and get access to the program without specifying any path prefix.
To test that the MISO packages are properly installed, verify that the two modules misopy and pysplicing are available from your Python environment:
>> import misopy >> import pysplicing
You should be able to import both of these packages without errors from the Python interpreter. The misopy package is the main MISO package, containing utilities for analyzing RNA-Seq data and plotting it (including sashimi_plot). The pysplicing package is an under-the-hood interface between Python and the statistical inference engine written in C.
To test that MISO can be run properly, run test_miso.py as shown below. These tests ensure that MISO can be run on a few genes/exons. The output of these tests can be ignored and the abbreviated version should be along the following lines:
$ python misopy/test_miso.py Testing conversion of SAM to BAM... ...output omitted... Computing Psi for 1 genes... ... ---------------------------------------------------------------------- Ran 3 tests in 68.000s OK
If you see errors prior to this, the most likely cause is that some dependency is not installed or not available in the path environment. For example, if samtools is installed but missing from the path (i.e. not accessible from the shell), that will cause errors in the test run.
To run MISO, a set of annotations (typically specified in GFF format) of the isoforms of alternative events must be provided and RNA-Seq reads (typically specified in SAM format). The GFF annotation is indexed into an efficient representation using a script provided by MISO. The SAM reads must be sorted and indexed into the BAM format (binary version of SAM) before they can be used with MISO.
The sections that follow describe in detail the file formats used for the annotation and for the reads and the specific steps that are required to run MISO. An example of a MISO pipeline for computing isoform expression estimates and detecting differentially expressed isoforms is given in Example MISO pipeline.
The figure below shows an overview of how to run MISO.
MISO can be run on either single or paired-end RNA-Seq data. Two general kinds of analyses are possible:
Exon-centric analyses are recommended for looking at alternative splicing at the level of individual splicing events, e.g. the inclusion levels of a particular skipped exon, or the use of a particular alternative splice site. In isoform-centric analyses the expression level of whole isoforms for genes are estimated (i.e. the expression of each individual isoform of a gene is estimated). Both of these analysis modes have merits and disadvantages. Exon-centric analyses are typically easier to interpret and validate experimentally, but do not always capture the complexity of a set related splicing events within a gene. Isoform-centric analyses capture more of this complexity, but are limited by the typically short length of RNA-Seq reads, and inaccuracies or incompleteness in the annotation of the gene’s isoforms. Estimates of isoform-level expression are also harder to validate in the lab using traditional techniques such as RT-PCR.
To run MISO, an annotation of the alternative splicing events or isoforms must be provided and a set of reads. Single or paired-end reads can be provided in the Spliced Alignment/Map (SAM) format (in its binary form, BAM) and the annotation can be in the GFF (version 3) format. The GFF format can be used to specify either whole mRNA isoforms of genes (which is the common use of the format) or to specify single alternative splicing events, as described in Alternative event annotations.
When run locally and not on a cluster, MISO will use multiple cores on the machine on which it is running. The number of cores/processors to be used by MISO is set through the settings file (see Visualizing RNA-Seq reads along isoforms with MISO estimates).
It is highly recommended to run MISO on a cluster when possible. Since each gene/exon can be treated as an independent inference problem, MISO is highly parallelizable, and the use of multiple computers can cut run times down by orders of magnitude.
MISO comes with basic functionality for running on a cluster, assuming that there’s a shared filesystem between all the nodes.
Alternative event annotations are available for the major classes of alternative splicing and alternative RNA processing events in the human (hg18, hg19), mouse (mm9) and Drosophila melanogaster genomes. The event types covered are:
For performing isoform-centric analyses, any gene models annotation can be used (e.g. from Ensembl, UCSC or RefSeq) as long as it is specified in the GFF3 format. As an example, we provide GFF3 annotations from Ensembl (which were converted from Ensembl’s GTF format to GFF3), available in Human/mouse gene models for isoform-centric analyses.
These annotations include GFF files (.gff3 extension) that can be used with MISO. The annotations for human and mouse were compiled as described in Wang et. al. (2008). Briefly, each splicing event was considered alternative if it was supported by several ESTs, and alternative tandem 3’ UTRs (TandemUTR events) were derived from PolyA DB.
A recent paper from Bin Tian’s group, Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing, annotated alternative cleavage and polyadenylation events in mouse tissues using the 3′READS method. The annotated events (TandemUTR and ALE) are available in GFF format here, courtesy of the Tian group:
Thanks to Wencheng Li and Bin Tian for making these annotations available.
Single alternative splicing events can be described in GFF format, using the convention described below. In the mouse and human genome event annotations in Alternative event annotations, alternative splicing events that produce two isoforms are described. For example, here is a skipped exon from the set of mouse skipped exons annotation:
chr1 SE gene 4772649 4775821 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-;Name=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:- chr1 SE mRNA 4772649 4775821 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:- chr1 SE mRNA 4772649 4775821 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.B;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:- chr1 SE exon 4775654 4775821 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A.up;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A chr1 SE exon 4774032 4774186 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A.se;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A chr1 SE exon 4772649 4772814 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A.dn;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.A chr1 SE exon 4775654 4775821 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.B.up;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.B chr1 SE exon 4772649 4772814 . - . ID=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.B.dn;Parent=chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-.B
The ID of the exon was chosen arbitrarily to be chr1:4775654:4775821:-@chr1:4774032:4774186:-@chr1:4772649:4772814:-. This name encodes the genomic coordinates of the upstream (5’) exon, the skipped exon, and the downstream (3’) exon of this alternative splicing event, separated by @ symbols. The two isoforms that result (encoded as mRNA entries in the file) are: (1) the isoform made up of the upstream exon, the skipped exon and the downstream exon (isoform where the exon is included), (2) the isoform made up of the upstream exon followed by the downstream exon (isoform where the exon is skipped.) Note that we only use the two flanking exons in this annotation, but one could modify the annotation to include arbitrarily many flanking exons from the transcripts. We chose to name the two mRNA entries as the coordinates of the exons that make up the mRNA followed by A for the first isoform, B for the second, though these naming conventions are arbitrary and do not have to be used. Similarly, the ID of each exon is encoded as its coordinate followed by up, se or dn to denote the upstream, skipped or downstream exons, respectively.
Below is a mapping of alternative events to genes, based on Ensembl annotation. These are tab-delimited files the first column (event_id) is the ID of the event from its GFF file and the second column (gene_id) corresponds to a comma-separated list of Ensembl identifiers for the gene(s) the event overlaps. If the event overlaps multiple genes (which could happen because multiple Ensembl identifiers are sometimes given to the same gene, or because the genes overlap and/or are contained within each other in the annotation), then multiple Ensembl identifiers will be listed. A mapping file is given for each event type (e.g. skipped exons, tandem 3’ UTRs, etc.) Events that cannot be mapped to genes are recorded as NA.
Note that in the above GFF annotations for events, only immediately flanking exons to an alternative event are considered. For example, for a skipped exon event, only the two exons that flank it are considered in the annotation. In the human/mouse genomes, this means that the two isoforms produced the event would be roughly ~400 nt and ~300 nt long. Modified annotations that use more of the exons present in the two isoforms might be more appropriate for use with long insert length paired-end libraries where the average insert is significantly longer than these isoform lengths. This in general will not affect isoform-centric analyses, where the entire mRNA transcript is used as the annotation to quantitate.
For isoform-centric analyses, annotations of gene models are needed, where whole mRNA isoforms are specified for each gene. Any annotation that is in GFF3 format can be used, e.g. annotations obtained from RefSeq, Ensembl, UCSC or other databases.
Annotations can be converted from UCSC table formats or from GTF format into GFF3 using the following procedure:
- Download your gene annotation (e.g. Ensembl genes, RefSeq) from UCSC Table Browser. For example, an Ensembl genes table ensGene.txt can be downloaded for the mm9 genome from UCSC goldenPath here (http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/ensGene.txt.gz).
- To convert this gene table into GFF3, use of these two ways: (a) use the script ucsc_table2gff3.pl (from biotoolbox) to convert the table directly into GFF3, or (b) use the UCSC utility genePredToGtf to convert the table into GTF format (instructions for this are described in http://genomewiki.ucsc.edu/index.php/Genes_in_gtf_or_gff_format) and then convert the GTF file into GFF3 using gtf2gff3.pl.
For convenience, we provide GFF3 annotations based on UCSC Table Browser’s version of Ensembl genes for the following genomes:
It is critical to note that Ensembl and UCSC have distinct chromosome naming conventions (see warning note below). However, Ensembl tables obtained from the UCSC Table Browser will follow the UCSC chromosome naming conventions, not Ensembl’s, which is why the UCSC-downloaded Ensembl annotations in the GFFs provided above contain chr headers. Ensembl annotations obtained directly from Ensembl (e.g. via Ensembl BioMart) will of course follow the Ensembl chromosome naming conventions, and not UCSC’s.
The UCSC and Ensembl databases name their chromosomes differently. By convention, UCSC chromosomes start with chr while Ensembl chromosome names do not. UCSC will call the fifth chromosome chr5 and Ensembl will call it 5. If your GFF annotation uses one convention, but your BAM reads are aligned to distinct headers, i.e. chromosome names following another convention, then none of your reads will map. For example, MISO will try to find all the regions on chr5 if your GFF annotation follows the UCSC convention and fail, since all your BAM alignments could be to a header named 5, or vice versa. In that case, lines such as these will appear in the MISO output:Cannot fetch reads in region: 5:1200-1500
We strongly recommend that you check that your BAM files follow the same headers naming convention as your GFF. If you run into a mismatch between your annotation’s chromosome headers and your BAM file headers, please make sure that the headers that you map to in the read mapping stage to are the same as the headers used in the GFF annotation for MISO. For example, if you use TopHat/Bowtie to map reads and your genome index was made with UCSC-style chr headers but you want to use Ensembl annotations for quantitations, the solution is to:
- Download your genome’s sequence files (*.fa, typically) from Ensembl,
- Make a new Bowtie index from these files (using bowtie-build), or the appropriate index for your read mapping program,
- Remap your reads against this new index. The resulting mapped SAM/BAM files can be used with the unmodified Ensembl GFF annotations we provide here, where the headers in the GFF are guaranteed to match the headers in the SAM/BAM files (both using Ensembl header conventions.)
For convenience, we also provide GFF3 annotations of gene models from Ensembl (release 65), which were simply converted from Ensembl’s GTF to GFF3 format and are otherwise identical to the Ensembl annotation.
When installing the MISO package, the set of scripts that make up to inteface to MISO will automatically be placed as an executables in your path, so that you can refer to these without modifying your shell environment. For example, if you install MISO using setup.py install globally, then a MISO script like run_events_analysis.py will become available on the path, and can be run as:
Or alternatively, as:
python run_events_analysis.py ...
Though the python prefix is not necessary. The path of the Python version on the system used to install MISO will automatically be invoked when running these scripts.
If you installed MISO using setup.py --prefix=~/ in your home directory or some other path, these scripts will typically be placed in a designated executables directory in your home directory, e.g. ~/bin/.
MISO can take as input RNA-Seq reads in the SAM format. SAM files are typically the output of read alignment programs, such as Bowtie or Tophat. The SAM format can represent both single and paired-end reads.
Files in SAM format must be converted to an indexed, sorted BAM file that contain headers. If you have an indexed, sorted BAM file you can move on to the next section.
The right BAM file can be created using samtools manually, or with the help of the wrapper script sam_to_bam.py that calls samtools. For example, to convert my_sample.sam to an indexed, sorted BAM file with headers specified in mm9_complete.fa.fai and output the result to the directory my_sample/, use:
python sam_to_bam.py --convert my_sample.sam my_sample/ --ref mm9_complete.fa.fai
The mm9_complete.fa.fai is a header file that contains the lengths of each chromosome in the genome used to create the my_sample.sam file. The .fai files can be created from the FASTA files of a genome using the faidx option of samtools (e.g. samtools faidx genome.fasta).
The sam_to_bam.py script will output a set of BAM files and their indices (my_sample.bam, my_sample.sorted.bam, my_sample.sorted.bam.bai) to my_sample/.
To quantitate alternative splicing, an annotation file must be provided that describes the isoforms whose expression should be estimated from data.
To estimate the expression level of whole mRNA transcripts, a GFF format containing a set of annotated transcripts for each gene can be used. This is the same GFF that can be given as input to Tophat in order to incorporate known genomic junctions, for example. These GFF files can be readily produced for any annotated genome (see Human/mouse gene models for isoform-centric analyses for Ensembl-based annotations of this form for human/mouse genomes.)
For exon-centric analyses, a GFF file can be used as well, except such a file will contain only the subsets of the isoforms produced by a particular splicing event – rather than whole transcripts – as described in GFF-based alternative events format. The GFF file chosen has to be indexed for efficient representation. A set of indexed, ready-to-use events is available for the mouse and human genomes.
To create an index for the GFF annotations, the index_gff.py script can be used as follows:
python index_gff.py --index SE.mm9.gff indexed/
This will index the GFF file SE.mm9.gff and output the result to the indexed/ directory. This indexed directory is then used as input to MISO.
The default configuration file for MISO is available in settings/miso_settings.txt. Skip to the next section if you’d like to use the default configuration file. The default file is:
[data] filter_results = True min_event_reads = 20 strand = fr-unstranded [cluster] cluster_command = qsub [sampler] burn_in = 500 lag = 10 num_iters = 5000 num_processors = 4
The [data] section contains parameters related to the way reads should be handled. These are:
The [cluster] section specifies parameters related to running MISO on a cluster of computers. These are:
Both long_queue_name and short_queue_name can be omitted from the configuration file, in which case jobs will be submitted to the cluster without specifying any queue. All parameters set in the [clusters] section are only relevant when MISO is used on a cluster.
The [sampler] section specifies internal parameters related to MISO’s sampling algorithm. Most users will not want to tweak these. The parameters are as follows:
The default settings of the sampling-related parameters in [sampler] was deliberately chosen to be conservative.
To run MISO to estimate the expression level of a set of annotated isoforms or events in GFF format, the run_events_analysis.py script is used with the --compute-genes-psi option. This produces distributions over “Psi” values (Ψ), which stand for “Percent Spliced In”. See Katz et. al. (2010) for a description of these values and how they are computed.
The --compute-genes-psi takes the following arguments:
--compute-genes-psi Compute Psi values for given GFF annotation. Expects two arguments: an indexed GFF directory with genes to process, and a sorted, indexed BAM file (with headers) to run on.
For example, to run on a single-end sample:
python run_events_analysis.py --compute-genes-psi mm9/pickled/SE data/my_sample.sorted.bam --output-dir my_sample_output/ --read-len 35
This will compute expression values for the isoforms described in the indexed GFF directory mm9/pickled/SE, based on the reads in data/my_sample.sorted.bam (a sorted, indexed BAM file) an output the results to the my_sample_output/ directory. Note that the mm9/pickled/SE directory in this case must be generated by the user, by running index_gff.py.
The --read-len option is necessary and specifies the length of the reads in the data (in this case, 35 nt.) The directory mm9/pickled/SE, which is available in the mouse splicing event annotations, specifies an indexed version of all the skipped exon (SE) events.
Note that that the headers for the indexed/sorted BAM file (the files with the .bam.bai extension) must be in the same directory as the read BAM file. In the above example, it means they must be in the data/ directory.
If reads were aligned to a set of splice junctions with an overhang constraint — i.e., a requirement that N or more bases from each side of the junction must be covered by the read — then this can be specified using the --overhang-len option:
--overhang-len Length of overhang constraints imposed on junctions.
To compute expression levels using paired-end reads, use the --paired-end option:
--paired-end Run in paired-end mode. Takes mean and standard deviation of insert length distribution.
The insert length distribution gives the range of sizes of fragments sequenced in the paired-end RNA-Seq run. This is used to assign reads to isoforms probabilistically. The insert length distribution can be computed by aligning read pairs to long, constitutive exons (like 3’ UTRs) and measuring the distance between the read mates. The mean and standard deviation of this distribution would then be given as arguments to --paired-end. For example, to run on a paired-end sample where the mean insert length is 250 and the standard deviation is 15, we would use: --paired-end 250 15 when calling run_events_analysis.py (in addition to the --compute-genes-psi option).
The --overhang-len option is not supported for paired-end reads.
We provide a set of utilities for computing and plotting the insert length distribution in paired-end RNA-Seq samples. The insert length distribution of a sample is computed by aligning the read pairs to long constitutive exons and then measuring the insert length of each pair. The set of insert lengths obtained this way form a distribution, and summary statistics of this distribution – like its mean and standard deviation – are used by MISO to assign read pairs to isoforms.
The utilities in exon_utils.py and pe_utils.py can be used to first get a set of long constitituve exons to map read pairs to, and second compute the insert length distribution and its statistics.
When computing the insert length distribution, it’s important to use exons that are significantly larger than the insert length that was selected for in preparing the RNA-Seq library. If the insert length selected for in preparing the RNA-Seq library was roughly 250-300 nt, we can use constitutive exons that are at least 1000 bases long, for example, so that both read pairs will map within the exon and not outside of it. The requirement that the exons be constitutive is to avoid errors in the insert length measurements that are caused by alternative splicing of the exon (which could alter the insert length.)
We first get all constitutive exons from a gene models GFF, like the Ensembl annotation of mouse genes (available here, Mus_musculus.NCBIM37.65.gff), we use exon_utils.py:
python exon_utils.py --get-const-exons Mus_musculus.NCBIM37.65.gff --min-exon-size 1000 --output-dir exons/
This will output a GFF file (named Mus_musculus.NCBIM37.65.min_1000.const_exons.gff) into the exons directory containing only constitutive exons that are at least 1000 bases long. This file can be used to compute the insert length distribution of all mouse RNA-Seq datasets. Exons here are defined as constitutive only if they occur in all annotated transcripts of a gene.
Now that we have a file containing the long constitutive exons, we can compute the insert length for any BAM file using the pe_utils.py utilities. The option --compute-insert-len takes a BAM file with the RNA-Seq sample and a GFF file containing the long constitutive exons. The insert length is defined simply as the distance between the start coordinate of the first mate in the read pair and the end coordinate of the second mate in the read pair.
To compute the insert length for sample.bam using the constitutive exons file we made above, run:
python pe_utils.py --compute-insert-len sample.bam Mus_musculus.NCBIM37.65.min_1000.const_exons.gff --output-dir insert-dist/
This will efficiently map the BAM reads to the exons in the GFF file using bedtools, and use the result to compute the insert lengths. The command requires the latest version of bedtools to be installed, where the tagBam utility was added the ability to output the interval that each read maps to in its BAM output file. The reads that aligned to these exons will be kept in a directory called bam2gff_Mus_musculus.NCBIM37.65.min_1000.const_exons.gff/ in the insert-dist directory.
This will generate an insert length distribution file (ending in .insert_len) that is tab-delimited with two columns, the first specifying the exon that the read pairs were aligned to, and the second specifying a comma-separated list of insert lengths obtained from aligning the read pairs to that exon. The number of entries in the file corresponds to the number of exons from the original GFF that were used in computing the insert length distribution.
The first line (header) of the the insert length file gives the mean, standard deviation and dispersion values for the distribution, for example:
This indicates that the mean of the insert length distribution was 129, the standard deviation was 12.1, dispersion was 1.1, and the number of read pairs used to estimate the distribution and compute these values was 862,148.
The dispersion constant d is defined as the standard deviation σ of the insert length distribution divided by the square root of its mean μ:
Intuitively, the dispersion constant d corresponds to how variable the insert length distribution is about its mean. The lower the d value is, the tighter/more precise the insert length distribution is in the RNA-Seq sample. The dispersion constant and an interpretation of its values is discussed in the MISO paper (see Analysis and design of RNA sequencing experiments for identifying isoform regulation).
The insert length distribution can be plotted using the .insert_len file using sashimi_plot.
To compute the insert length by simple pairing of read mates by their ID (assuming that mates belonging to a pair have the same read ID in the BAM), explicitly ignoring the relevant BAM fields specifying whether or not the reads are properly paired, use the --no-bam-filter option.
The option --compute-insert-len can take a comma-separated list of BAM filenames in case you want to compute the insert length for multiple samples in one command. The --compute-insert-len option of pe_utils by default uses only exons from the GFF file that are 500 bases or longer. This can be tweaked by passing pe_utils the optional --min-exon-size N argument, which will only use exons of size N or longer. In our example, we used a constitutive exons GFF file that contained only exons that are 100 bp or longer, so the default settings will consider all exons in that file.
To increase efficiency, a prefiltering option was added in release 0.4.7. When --prefilter is given to run_events_analysis.py, MISO will calculate the number of reads in the input BAM mapping to each event in the input GFF. Events that do not meet the read coverage thresholds (as set in the configuration file) will be removed from the run. This feature requires the Bedtools utility tagBam to be installed and available on path. The call to tagBam introduces a startup cost per BAM file, but could in many cases save computation time, since events low coverage events will not processed or distributed as jobs to nodes when running on the cluster. From run_events_analysis.py --help:
--prefilter Prefilter events based on coverage. If given as argument, run will begin by mapping BAM reads to event regions (using bedtools), and omit events that do not meet coverage criteria from the run. By default, turned off. Note that events that do not meet the coverage criteria will not be processed regardless, but --prefilter simply does this filtering step at the start of the run, potentially saving computation time so that low coverage events will not be processed or distributed to jobs if MISO is run on a cluster. This options requires bedtools to be installed and available on path.
Running MISO on a cluster is highly recommended. Since each gene or alternative splicing event can be treated independently, the computation of expression levels is easily parallelized on a cluster. This can reduce run times significantly, from days to hours on typical data sets. To run MISO on the cluster, the following options can be used:
MISO will use the cluster submission and queue settings described in the settings file to submit jobs (see Visualizing RNA-Seq reads along isoforms with MISO estimates). An example of a MISO pipeline for computing isoform expression estimates and detecting differentially expressed isoforms is given in Example MISO pipeline.
To use MISO on Sun Grid Engine, the following options to run_events_analysis.py can be used:
Thanks to Michael Lovci for this feature.
When running MISO, through run_events_analysis.py --compute-genes-psi, the raw output will be a set of posterior distributions over Ψ values. Each exon or gene, depending on whether the analysis is exon or isoform-centric, will have its own file containing posterior samples from the distribution over Ψ.
Most users will never need to access these raw distributions and can simply summarize this raw output as described in Summarizing MISO output, or perform a comparison between a set of samples as described in Detecting differentially expressed isoforms.
Each gene or exon will have a corresponding distribution file (ending in .miso), split into subdirectories by chromosome. The resulting directory structure might be:
my_sample_output/ chr1/ chr2/ ... chrX/
The distribution file for an event, e.g. chr1/my_exon.miso, will contain tab-separated columns that specify the sampled Ψ values and the log score under the MISO model for each sample, e.g.:
#isoforms=['TandemUTRCore_TandemUTRExt','TandemUTRCore'] exon_lens=('TandemUTRCore',1986),('TandemUTRExt',1021) iters=5000 burn_in=500 lag=10 percent_accept=92.90 proposal_type=drift sampled_psi log_score 0.0691,0.9309 -989.1296 0.0692,0.9308 -1002.1173 0.0595,0.9405 -994.5029 0.0587,0.9413 -988.9871 0.0539,0.9461 -980.4465 0.0571,0.9429 -986.1921 0.0482,0.9518 -980.2666 0.0392,0.9608 -999.4088
The header line (beginning with #) gives information about the gene/event in the file, like the names of the isoforms in the annotation and the length of the exons, as well as values of internal MISO parameters (like burn-in or lag, described in Visualizing RNA-Seq reads along isoforms with MISO estimates).
The first column (sampled_psi) lists the Ψ value sampled and the second column (log_score) gives the log score of that sample under the MISO model. The Ψ value is comma-separated and will have as many entries as there are isoforms. In the above file, there are two isoforms, and so two entries in the Ψ column that sum to 1.
After raw MISO output is summarized and all pairwise comparisons between samples have been made, the raw output can be compressed (see Compressing raw MISO output.)
To summarize MISO output and obtain confidence intervals (CIs) for Ψ values, the run_miso.py --summarize-samples option can be used:
--summarize-samples Compute summary statistics of the given set of samples. Expects a directory with MISO output and a directory to output summary file to.
For example, to summarize the MISO output in my_sample_output/, run:
python run_miso.py --summarize-samples my_sample_output/ summary_output/
This will create a subdirectory summary_output/summary/ with a file my_sample_output.miso_summary that summarizes all the events in the sample in my_sample_output/.
The summary file is a tab-separated format containing the following columns:
Starting with release misopy-0.4.1, three additional columns are outputted with information about the gene/event (which are read from the input GFF annotation):
When one of those fields is not available (e.g. strand), NA is outputted in its place.
Note that miso_posterior_mean is an estimate of Ψ and [ci_low, ci_high] are the 95% confidence limits of that estimate.
Once MISO output has been computed for two samples or more, pairwise comparison of differential isoform expression can be computed. To test if an isoform or exon is differentially expressed between between samples, the Bayes factor is computed for the exon, which represents the weight of the evidence in the data in favor of differential expression versus not. For example, a Bayes factor of 2 would mean that the isoform/exon is two times more likely to be differentially expressed than not. The computation of Bayes factors is described in detail in Katz et. al. (2010).
To compute Bayes factors, the run_miso.py --compare-samples option can be used. For example:
python run_miso.py --compare-samples control/ knockdown/ comparisons/
This would compare the MISO output in control/ to knowndown/, and output the result to the directory comparisons/control_vs_knockdown. Note that “vs” is used to denote sample comparisons. The output format of this comparison is described below.
The main output of --compare-samples will be in:
The Bayes factors file (ending in .miso_bf) will be stored in the bayes-factors/ directory generated by the --compare-samples option. The Bayes factor file is a tab-separated format containing the following columns:
If MISO is run on an annotation that specifies more than two isoforms per event/gene, then the fields for the Ψ estimates become comma-separated and give the estimates for each isoform. For example, in the case of three isoforms, sample1_ci_low would be a comma-separated list of the lower bounds of the first, second and third isoform, and sample1_ci_high would be a comma-separated list of the upper bounds of the first, second and third isoform, and similarly for the fields involving the other sample.
Starting with release misopy-0.4.1, three additional columns are outputted with information about the gene/event (which are read from the input GFF annotation):
When one of those fields is not available (e.g. strand), NA is outputted in its place.
Once all the samples have been summarized and all pairwise comparisons between samples have been made (as described in Summarizing MISO output and Detecting differentially expressed isoforms), the directories containing the raw MISO output files (files ending in .miso) can either be deleted or compressed for later use.
To compress directories containing raw MISO output files, a utility (currently only available in GitHub repository) called miso_zip.py can be used. For example, to compress the directory miso_output/ into and name the result mydata.misozip, use:
python miso_zip.py --compress mydata.misozip miso_output/
(Note that compressed MISO files must end in .misozip.) miso_zip will take as argument any directory containing *.miso files (either directory within it or within its subdirectories) and collapse all the *.miso files into a portable SQLite database, split by chromosome. Each chromosome directory will be transformed into a .miso_db file, which can be read in a Python-independent way through any SQLite interface.
This compression reduces the number of files dramatically, since each sample gets one SQLite database per chromosome. The resulting directory structure is then compressed further using standard zip compression into a single zip file (in this case named mydata.zip).
The compressed .misozip files can be uncompressed using miso_zip as follows:
python miso_zip.py --uncompress mydata.misozip uncompressed/
This will uncompress mydata.misozip and output its contents into the directory uncompressed/.
If you would like to access the collapsed SQLite representation of the directory without retrieving the original *.miso files, all you need to do is unzip the .misozip file using the standard unzip utility:
This will give the original contents of the compressed directory except the *.miso files, which will now appear as *.miso_db (SQLite databases) arranged by chromosome for each sample.
Note that miso_zip.py will not delete the directory being compressed. The directory can be deleted manually after compression is done.
Below is an example of a MISO pipeline, where Ψ values for a set of alternative events are computed (with confidence intervals) for a pair of samples called “control” and “knockdown”. A samples comparison is performed to detect differentially expressed isoforms between the samples.
## Run MISO on a pair of paired-end sample (with insert length distribution with mean 250, ## standard deviation 15) using the mouse genome skipped exon annotations using the ## the cluster # Compute Psi values for control sample python run_events_analysis.py --compute-genes-psi mm9/pickled/SE data/control.bam --output-dir SE/control/ --read-len 35 --paired-end 250 15 --use-cluster # Compute Psi values for knockdown sample python run_events_analysis.py --compute-genes-psi mm9/pickled/SE data/knockdown.bam --output-dir SE/knockdown/ --read-len 35 --paired-end 250 15 --use-cluster ## Summarize the output (only run this once --compute-genes-psi finished!) ## This will create a "summary" directory in SE/control/ and in SE/knockdown/ python run_miso.py --summarize-samples SE/control/ SE/control/ python run_miso.py --summarize-samples SE/knockdown/ SE/knockdown/ ## Detect differentially expressed isoforms between "control" and "knockdown" ## This will compute Bayes factors and delta Psi values between the samples ## and place the results in the directory SE/comparisons/control_vs_knockdown python run_miso.py --compare-samples SE/control/ SE/knockdown/ SE/comparisons/
Once MISO runs are completed and pairwise comparisons between your samples have been made to compute Bayes factors (using --compare-samples), the resulting files can be interpreted to detect differential events. For each pairwise comparison, we will have a .miso_bf file which contains the Ψ values of each event in both samples along with confidence intervals (assuming the event is detectable in both), the Δ Ψ values, the Bayes factor, and other useful information about the read counts used to compute these values.
MISO comes with several utilities (such as filter_events.py, described in Filtering differentially expressed events) for helping with the interpretation and analysis of these output files. This section serves as a brief guide the MISO output files, the utilities for analyzing them, and to various caveats in interpreting the output.
A first-pass filter for detecting differentially changing events can apply a Bayes factor cutoff along with a Δ Ψ cutoff, using the bayes_factor and diff fields of the samples comparison output file. Section Filtering differentially expressed events describes a utility that will apply these filters for exon-centric analyses.
It is critical to note that the default MISO settings were intentionally set to be highly inclusive, and therefore do not impose any stringent coverage criteria on the events for which Ψ values and Bayes factors are computed.
By default, MISO requires only 20 reads to be present in the locus/event of interest. For many events, only a tiny fraction of these reads might be in informative regions – like junctions or regions unique to one of the isoforms – and so the expression levels of these events might not be reliably estimated. It is important to use the information provided by MISO about the raw number of read counts used in estimating the expression levels of each event/gene.
In addition to Ψ values, confidence intervals and Bayes factors, the output files of MISO (such as summary files or sample comparison files) also contain the number of read counts informative about each isoform. The relevant read counts are given in the counts field of the .miso, .summary, .miso_bf files – its format is described in detail in Summary file output format). For the two-isoform case in exon-centric analyses, the counts field general format is:
where X, Y, Z, L are integer counts corresponding to the number of reads in each of these categories. Class (1,0) are reads consistent with the first isoform in the annotation but not the second, class (0,1) are reads consistent with the second but not the first, class (1,1) are consistent with both isoforms, and reads in (0,0) are consistent with neither. These are illustrated graphically in the figure below for alternatively skipped exon event:
The class (0,0) denotes reads that were incompatible with either isoform, either because they didn’t match these isoforms or were otherwise unusable (e.g. they were part of a read-pair where one end was unmappable, in the case of paired-end reads.) Reads in this class are not used by MISO – they are only reported to indicate that they were discarded.
Events that meet the very minimal default coverage criteria do not necessarily have sufficient read support to be considered alternatively spliced in your RNA-Seq samples.
In practice, events that might not be alternatively spliced in the samples of interest meet the default minimal read coverage criteria, although their read class counts imply that they are not reliable estimates. Such a case might be an alternative exon in the input annotation to MISO that has, for example, 19 reads in the exon upstream of the skipped exon, and 1 read in the exon downstream of the skipped exon. This event would have a counts signature of: (1,1):20,(1,0):0,(0,1):0. The (1,0) and (0,1) reads, which are most informative about the relative inclusion levels of the exon, are both 0, so unless these are non-zero in other samples in the dataset, the estimate of the inclusion levels of this exon will not be reliable.
For this reason, coverage filters must be imposed by the user when post-processing MISO output. An example of such a filter is given below for exon-centric analyses.
A commonly-used coverage filter in exon-centric (two-isoform) analyses requires the following minimal counts criteria to be met:X + Y >= N, Y >= 1
in at least one of the RNA-Seq samples processed by MISO, where X, Y are the number of reads in the (1,0) and (0,1) read classes, respectively, and N is an arbitrary but sizeable number (e.g. 10 or 20). This filter requires that the sum of inclusion and exclusion-supporting reads be greater than or equal to N, and that the read class supporting exclusion is non-zero in at least one of the samples. This guarantees that at least some of the reads are informative about the inclusive or exclusive isoform. Note that this filter does not guarantee that there will be junction evidence for the inclusion isoform, but does guarantee that there will be a junction evidence for skipping of the exon.
To increase power to detect differential events, coverage filters should be applied in aggregate across all RNA-Seq samples that were processed by MISO. To detect switch-like exons that are fully included in one sample and fully excluded in another, we should require the number of exon exclusion supporting reads be non-zero in at least one of the samples – or summed across samples – rather than in every sample. This filter can be easily applied by parsing the counts field in the samples comparison (.miso_bf) file produced by MISO for each pairwise comparison.
For isoform-centric analyses where the number of read classes is considerably larger, MISO will output the counts in the same format. For a gene with 3 isoforms, we can have the read classes (1,0,0),(0,1,0),(0,0,1),...,(1,1,1) and other filters can be applied, depending on the goal of the analysis, to select genes that have sufficient coverage to be reliably estimated.
Important information is contained in the confidence intervals outputted by MISO for each estimate of Ψ. The width of the interval corresponds to our confidence in the estimate of the Ψ value, and so the larger the confidence width is, the less reliable the estimate. As expected, the width of the confidence interval will depend on the number of informative reads, such as reads crossing junctions or reads in regions that uniquely identify the isoform.
The figure on the right shows two posterior distributions over Ψ for the same alternative splicing event in two samples, where the exon had low coverage (i.e. small number of reads) in both. The posterior distribution is plotted as a black histogram, whose mean is indicated by the red vertical line and 95% confidence intervals shown by dotted grey vertical lines. The actual Ψ values and the confidence interval bounds are given to the right of the distribution. In the top distribution, the confidence interval width is .64 (since .88 - .24 = .64) which is extremely wide. The distribution’s probable range spans from 20 to nearly 90%, far too broad to be useful. In the bottom distribution, the confidence intervals are narrower, though still considerable. Because of the large amount of uncertainty in the top distribution, we cannot confidently conclude that the true Ψ value is likely to be different between the two. Although the difference of means between the distributions is large (.53 - .2 = .33), the confidence intervals overlap considerably as a result of the uncertainty in the top distribution. In practice, requiring no overlap at all between the confidence intervals of two samples is too restrictive, but the width of each estimate’s confidence interval and the relationship between the intervals should be kept in mind when interpreting the results.
Given a MISO Bayes factor comparison file for two-isoform events, events can be filtered based on their coverage or magnitude of change. The filter_events.py script allows filtering of events, based on the following criteria:
For example, to filter the file control.miso_bf to contain only events with: (a) at least 1 inclusion read, (b) 1 exclusion read, such that (c) the sum of inclusion and exclusion reads is at least 10, and (d) the Δ Ψ is at least 0.20 and (e) the Bayes factor is at least 10, and (a)-(e) are true in one of the samples, use the following command:
python filter_events.py --filter control.miso_bf --num-inc 1 --num-exc 1 --num-sum-inc-exc 10 --delta-psi 0.20 --bayes-factor 10 --output-dir filtered/
This will output a filtered Bayes factor file, filtered/control.miso_bf.filtered that contains only events meeting the above criteria.
MISO comes with a built-in utility, sashimi_plot, for visualizing its output and for plotting raw RNA-Seq read densities along exons and junctions.
- MISO is passed a BAM file that is not sorted and indexed. BAM files must be sorted and indexed to allow MISO random access to regions in the BAM.
- The BAM file was indexed, but the .bai file (which holds the index) is not present in the same directory as the .bam file passed to MISO. Indexing the file and ensuring that its corresponding index file is in the same directory will fix the problem. Related issue: the BAM file and the BAM file index mismatch due to capitalization differences in the filenames. For example, your BAM file is reads.bam, but the index is reads.BAM.BAI (note capitalization differences in extension). This will prevent MISO from pairing the index with the BAM file. The index for reads.bam must be instead named reads.bam.bai
- The chromosome headers of the input BAM file and of your indexed annotation mismatch (e.g. annotation uses UCSC chr style headers, while BAM file uses Ensembl chromosome headers.)
- MISO was passed the wrong read length: for example, the reads in your BAM file are of length 50 but you passed a different read length in the --read-len argument. MISO requires the correct read length to be passed.
The ultimate way to test if your BAM file can be accessed by MISO is to pick a region from the GFF annotation that you’re passing to MISO that you know should contain some reads, e.g. chr1:100-300 and try to access this via samtools:
samtools view reads.bam chr1:100-300
If samtools cannot access the reads in that region, MISO will not be able to either. Failure to access reads in the region is typically caused by one of the above issues. (back)
The SEC format is deprecated - we recommend using BAM files along with GFF annotation files instead, as that format is more flexible and standard, and allows representation of both individual alternative splicing events (for exon-centric) and whole mRNA isoforms (for isoform-centric analyses).
The Single-end Event Counts (SEC) format can be used to quantitate individual alternative splicing events (exon-centric analyses) using single-end RNA-Seq data sets. This format does not support paired-end information and is best suited for cases where individual events are quantitated. The format currently supports skipped exons (SE), alternative 3’ UTRs (TandemUTR), and retained introns (RI). The format is described below.
The SEC format is a two-column tab-separated format, where the first column identifies the coordinates of the alternative events, and the second gives a set of counts. The elements in each field as always separated by semicolons (;).
For skipped exons, the SEC format is:
where the first column specifies the coordinates as follows:
and the second column specifies the counts for the skipped exon event:
The reads corresponding to each set of counts are shown in the figure above.
An example SE event is:
For alternative tandem 3’ UTRs, the SEC format is:
where the first column specifies the coordinates of the core and extension regions of the TandemUTR event as follows:
and the second column specifies the counts in the regions:
ext_count: number of reads in the extension region,
core_count: number of reads in the core region
The coordinates of the event are given in the order core, extension but the counts are given in the opposite order of extension count followed by core count.
The reads corresponding to each count are shown in the figure above.
An example TandemUTR event is:
For retained introns, the SEC format is:
where the first column specifies the coordinates of the retained intron and its flanking exons as follows:
and the second column specifies the counts in the regions:
An example RI event is:
The reads corresponding to each count are shown in the figure above.
To run MISO on a SEC format, the run_events_analysis.py --compute-events-psi script is used:
--compute-events-psi Compute Psi values for all events. Expects two arguments: a set of labels and a set of filenames with associated read counts.
The first argument to --compute-events-psi is a comma-separated set of sample labels and the second is a comma-separated set of counts filenames. For instance, --compute-events-psi control,knockdown control.counts,knockdown.counts would run MISO on the SEC files control.counts and knockdown.counts, labeling the output directories control and knockdown, respectively. When running on a single sample, the comma notation can be dropped (e.g. --compute-events-psi control control.counts).
To use --compute-events-psi, the type of event must be specified through the --event-type option, which can be either SE (skipped exons), TandemUTR (alt. tandem UTRs) or RI (retained introns.) The sequenced read length must be specified with --read-len. Optionally, the overhang length imposed on junction reads during alignment can be specified with --overhang-len.
For example, the following command would run MISO on a set of skipped exons in the SEC file control.counts in an experiment where the reads are 35 nt long and an overhang length of 4 was placed on junction reads during alignment:
python run_events_analysis.py --compute-events-psi control control.counts --event-type SE --read-len 35 --overhang-len 4 --output-dir control
This will create a directory called SE/control/ with MISO output for each event in control.counts that meets the default read counts filter.
Rather than estimating the expression level of a single alternatively spliced exon (“exon-centric”), or of each transcript belonging to a gene (“isoform-centric”), one can quantify the levels of multiple isoforms produced by several nearby alternative splicing events. This is called local multi-isoform quantitation. These isoforms can be encoded in the GFF format as for the case of alternative splicing events that produce two isoforms, described in GFF-based alternative events format.
Section under construction
Thanks to the following users for their feedback/comments on MISO and for suggesting many improvements, or for creating useful software used by MISO:
The Python-only version of MISO is now deprecated – we recommend using fastmiso.
This version requires:
MISO has not been tested with Python 3.x. Once these libraries are available, the Python-only version of MISO can be used.
To get the most up to date version of the Python-only MISO, we recommend checking out the latest version of the dev branch from our GitHub repository.