Introduction
MicroExonator is a fully-integrated computational pipeline that allows for systematic de novo discovery and quantification of microexons using raw RNA-seq data for any organism with a gene annotation. Compared to other available methods MicroExonator is more sensitive for discovering smaller microexons and it provides higher specificity for all lengths. Moreover, MicroExonator provides integrated downstream comparative analysis between cell types or tissues using Whippet (Sterne-Weiler et al. 2018). As a proof of principle MicroExonator identified X novel microexons in Y RNA-seq samples from mouse early development to provide a systematic characterization based on time and tissue specificity.
Installation
Start by cloning MicroExonator
git clone https://github.com/hemberg-lab/MicroExonator
Install Miniconda 3
wget
chmod +x Miniconda3-latest-Linux-x86_64.sh
./Miniconda3-latest-Linux-x86_64.sh
Finally, create an enviroment to run snakemake
conda create -n snakemake_env -c bioconda -c conda-forge snakemake
Configuration
Before running MicroExonator you need to have certain files in the MicroExonator/
directory. By default, all the downloaded files and the results will be stored in the MicroExonator/
folder which means that several GBs of disk space may be required. First, you need to create a config.yaml
, which should contain the path of the input files and certain paramethers. The format for the config.yaml
file is shown below:
Genome_fasta : /path/to/Genome.fa
Gene_anontation_bed12 : /path/to/ensembl.bed12
GT_AG_U2_5 : /path/to/GT_AG_U2_5.good.matrix
GT_AG_U2_3 : /path/to/GT_AG_U2_3.good.matrix
conservation_bigwig : /path/to/conservation.bw
working_directory : /path/to/MicroExonator/
ME_DB : /path/to/ME_DB.bed
ME_len : 30
Optimize_hard_drive : T
Here:
Genome_fasta
is a multifasta file containg the cromosomes.Gene_anontation_bed12
is a BED file containing the transcript annotation. A collection of these files can be found at UCSC genome browser.GT_AG_U2_5
andGT_AG_U2_5
are splice site PWMs that can come from SpliceRack (please remove the first lines so it looks like the files that we have in/PWM
). If you do not have the PWMs for the species you are interested in, you can leave it asNA
and MicroExonator will generate the PWM internally based on annotated splice sites.conservation_bigwig
is a bigwig file containing genome-wide conservation scores generated by Pylop or PhastCons, which can be downloaded from UCSC genome browser for some species. If you do not have aconservation_bigwig
file, you need to create an bigwig which has the value 0 for every position in your genome assembly.working_directory
is the path to the MicroExonator folder that you are working withME_len
is microexon maximum lenght. The default value is 30.ME_DB
is a path to a known Microexon database such as VAST DB (is this optional or no?)Optimize_hard_drive
can be set asT
orF
(true of false). When it is set asF
, fastq files will be downloaded or copied only once atFASTQ/
directory. This can be inconvenient when you are analysing large amout of data (for instance when your input data is larger than 1TB), because the copy will not be deleted until MicroExonator finish completely. WhenOptimize_hard_drive
is set asT
instead, two independet local copies will be generated for the discovery and quantification moduled. As these are temporary copies, every copied fastq file will be deleted as soon as is mapped to the splice junction tags, which means the disk space usage will be set to the minimum while MicroExonator is runing.
To imput the RNA-seq data, you need to either create a design.tsv
(for fastq.gz files that are stored locally) and/or NCBI_accession_list.txt
(for SRA accession names) which are automatically downloaded. You can find examples of these files inside the Examples folder.
Finnaly, if you are working on a high performace cluster, then it is very likely that you need to submit jobs to queueing systems such as lsf, qsub, SLURM, etc. To make MicroExonator work with these queueing systems, you need to create a cluster.json
file. We currently provide in the Examples folder a cluster.json
file to run MicroExonator with lsf. To adapt MicroExonator to other quequing systems please see the SnakeMake documentation.
Running
We highly recommend creating a screen before running MicroExonator
screen -S session_name #choose a meaning full name to you
To activate snakemake enviroment
conda activate snakemake_env
Then run
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs}
Before running it is recommended to check if SnakeMake can corretly generate all the steps given your input. To do this you can carry out a dry-run using the -np
parameter:
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs} -np
The dry-run will display all the steps and commands that will be excecuted. If the dry-run cannot be initiated, make sure that you are running MicroExonator from inside the folder you cloned from this repository. Also make sure you have the right configuration inside config.yaml
.
Notice that you should use --cluster
only if you are working in a computer cluster that uses a queuing systems. We provide an example of cluster.json
to work with lsf and in that case the cluster system params should be "bsub -n {cluster.nCPUs} -R {cluster.resources} -c {cluster.tCPU} -G {cluster.Group} -q {cluster.queue} -o {cluster.output} -e {cluster.error} -M {cluster.memory}"
. The number of parallel jobs can be a positive integer, the appropriate value depends on the capacity of your machine but for most users a value between 5 and 50 is appropriate.
If you want to process a large dataset, we recommend to set Optimize_hard_drive
as T
. If you tho this, you can also run the pipeline in two steps:
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs} discovery
Once the pipeline finish the discovery module, it can be resumed any time later as:
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs} quant
If you do not have space to have a let MicroExonator to have a complete copy of the data you want to process, you can limmit the number of jobs. For instance if you limit the number of jobs to 20, you can make sure no more than 20 fastq files will be stored at FASTQ/
directory (if Optimize_hard_drive
is set as T
)
If you are working remotelly, the connection is likely to die before MicroExonator finish. However, as long as you are working within an screen, loggin off will not kill snakemake. To list your active screens you can do:
screen -ls
To reattached and detach screens just use:
screen -r session_name # only detached screen can be reattached
screen -d session_name
Alternative splicing analyses
Microexonator can couple the annotation and quantification of microexon with other downstream tools to asses alternative splicing changing. We have incorporated whippet into a downstream snakemake workflow that can directly use the microexon annotation and quantification files to assess differential inclusion of microexons between multiple conditions. For this purpose we recomend to install a custumised conda enviroment that has snkamemake and julia 0.6.1 installed. This can be don by using the following command from inside a MicroExonator folder:
conda env create -f Whippet/julia_0.6.1.yaml
Where Whippet/julia_0.6.1.yam
is yaml file with the recipe to create a stable environment with julia 0.6.1 and snakemake.
Then, activate the newly created enviroment:
source activate julia_0.6.1
Enter julia's interactive mode:
julia
Install Whippet:
Pkg.add("Whippet")
Exit interactive julia session (control + d
) and find Whippet's binary folder, that should be inside of your miniconda environment folder. Once you find the path to this folder, add it to config.yaml
writing the following lines:
whippet_bin_folder : path/to/miniconda/envs/julia_0.6.1/share/julia/site/v0.6/Whippet/bin
Gene_anontation_GTF : path/to/gene_annotation.gtf
whippet_delta:
Control_vs_TreatmentA :
A : SRR309144,SRR309143,SRR309142,SRR309141
B : SRR309136,SRR309135,SRR309134,SRR309133
Control_vs_TreatmentB :
A : SRR309140,SRR309139
B : SRR309138,SRR309137
Where whippet_delta
correspond to a dictionary that has all the comparions betweeen samples that you would like to do. Each comparison can have an arbitrary name (here as Control_vs_TreatmentA
and Control_vs_TreatmentB
). For each comparison two groups of samples are compared; A
and B
. These correspond to comma-separated list of the replicates for the conditions you are interested to compare.
Then we recomend to do a dry-run
to check that all the inputs are in place:
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs} differential_inclusion -np
Important: If you arlready run MicroExonator quantification and discovery, and now you want to perform the these downstream alternative splicing analyses, you might need to skip microexonator steps to avoid them be re-run. To only perfom downstream alternative splicing analyses, you will need to include these extra key in config.yaml:
downstream_only : T
If you do this, you will schedule processes that are related with whippet
indexing, quantification and differential inclusion. For example if you have a total of 6 samples, you will see something like this:
Job counts:
count jobs
6 ME_psi_to_quant
1 delta_ME_from_MicroExonator
1 delta_ME_from_whippet
1 differential_inclusion
6 download_fastq
1 get_GTF
6 gzip_ME_psi_to_quant
1 whippet_delta
1 whippet_delta_ME
1 whippet_index
6 whippet_quant
31
After you are sure everthing is in place, you can sumbit the runnig command:
snakemake -s MicroExonator.skm --cluster-config cluster.json --cluster {cluster system params} --use-conda -k -j {number of parallel jobs} differential_inclusion
Using MicroExonator on single-cell RNAseq data
MicroExonator can also allow you to identify microexons and identify patterns of alternative splicing changes using whippet in scRNAseq data. Importantly, we have only applied MicroExonator to full-length scRNAseq data (SMART-seq2 in particular). This is because full-length scRNAseq data provides more information to resolve splicing changes as well as due to the accuracy of bulk RNAseq tools for alternative splicing identification being benchmarked by our group before and showed promising results.
Troubleshooting
Check that every necesary key is writen correctly at config.yaml
. Also, when you provide the gene annotation files, ensure that all the gene chromosomes are available at the genome fasta that you are providing. Any missing chromosome, will generate errors at different parts of the workflow.
After the release of conda 4.4 some additional configuration might be required to work with snakemake worflow that have fix conda enviroments, particulaly there might be some inteference of some conda enviroments when that pipeline call is done from an screen interference with screen comand. Thus, we recomend modify your ~/.bashrc
file and add the following line:
. /path/to/miniconda/etc/profile.d/conda.sh
Where /path/to/miniconda
is the directory where you installed Miniconda, which is at your home directory by default, but during Miniconda instalation can be set at any directory.
If you have any errors while you are running MicroExonator is useful to read the logs that are reported by the queuing system. Some errors may occur because when not enough memory has been allocated for a given step. Resources for each step can be this can be configured inside cluster.json
(check example file at MicroExonator/Examples/Cluster_config/lsf/)
When some step fails, you can always resume MicroExonator run to avoid starting from scratch. To do this is important to check that there are not interrupted files. If snakemake finish or is manually interrupted (only once) it will flag interrupted files and delete them before you resume the pipeline. However, when downloading steps are interrupted, snakemake will not check whether the fastq files inside FASTQ/
are complete or not. Thus, if you are not sure if fastq files inside FASTQ
are complete, please delete all fastq files inside FASTQ/
before running MicroExonator again. Once you do this, you run MicroExonator again as dry-mode (with -np
), some jobs might be already completed, so you might have less remaining steps to finish the analysis.
Contact
For questions, ideas, feature requests and potential bug reports please contact gp7@sanger.ac.uk.