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Running SQANTI3 rescue
As of version 5.1, a third module has been added to the SQANTI3 workflow for transcriptome characterization and quality control. The SQANTI3 rescue algorithm is designed to be run after transcriptome filtering and intends to use the long read transcript evidence provided by discarded isoforms (i.e. artifacts) to avoid losing transcripts/genes that are detected as expressed, but whose start/end/junctions could not be confidently validated using orthogonal data. In particular, during the rescue, SQANTI3 will try to confidently assign each discarded artifact to the best matching reference transcript. As a result, SQANTI3 rescue will generate an expanded transcriptome GTF including a set of reference transcripts as well as the long read-defined isoforms that passed the filter.
Similarly to the SQANTI3 filter, the SQANTI3 rescue is designed as a
dual implementation, depending on whether the rules or the machine learning filter was previously run. Therefore, the sqanti3_rescue.py
script requires a flag to be provided to activate either the ml
or rules
specific rescue.
usage: sqanti3_rescue.py [-h] {ml,rules} ...
Rescue artifacts discarded by the SQANTI3 filter, i.e. find closest match for
the artifacts in the reference transcriptome and add them to the
transcriptome.
positional arguments:
{ml,rules}
optional arguments:
-h, --help show this help message and exit
To be completed: further justify why transcript rescue is required after filtering.
TBC: step-by-step descrption of the rescue strategy implemented in SQANTI3. Decision tree for rescue.
These are the arguments accepted by sqanti3_rescue.py rules
:
usage: sqanti3_rescue.py rules [-h] [--isoforms ISOFORMS] [--gtf GTF] [-g REFGTF]
[-f REFGENOME] [-k REFCLASSIF]
[-e {all,fsm,none}] [-o OUTPUT] [-d DIR]
[--skip_report] [-v] [-j JSON]
sqanti_filter_classif
Rescue for rules-filtered transcriptomes.
positional arguments:
sqanti_filter_classif
SQANTI filter (ML or rules) output classification file.
optional arguments:
-h, --help show this help message and exit
--isoforms ISOFORMS FASTA file output by SQANTI3 QC (*_corrected.fasta), i.e. the full long read transcriptome.
--gtf GTF GTF file output by SQANTI3 filter (*.filtered.gtf).
-g REFGTF, --refGTF REFGTF
Full path to reference transcriptome GTF used when running SQANTI3 QC.
-f REFGENOME, --refGenome REFGENOME
Full path to reference genome FASTA used when running SQANTI3 QC.
-k REFCLASSIF, --refClassif REFCLASSIF
Full path to the classification file obtained when running SQANTI3 QC on the reference
transcriptome.
-e {all,fsm,none}, --rescue_mono_exonic {all,fsm,none}
Whether or not to include mono-exonic artifacts in the rescue. Options include: none, fsm and all
(default).
-o OUTPUT, --output OUTPUT
Prefix for output files.
-d DIR, --dir DIR Directory for output files. Default: Directory where the script was run.
--skip_report Skip creation of a report about the filtering
-v, --version Display program version number.
-j JSON, --json JSON Full path to the JSON file including the rules used when running the SQANTI3 rules filter.
These are the arguments accepted by sqanti3_rescue.py rules
:
usage: sqanti3_rescue.py ml [-h] [--isoforms ISOFORMS] [--gtf GTF] [-g REFGTF]
[-f REFGENOME] [-k REFCLASSIF]
[-e {all,fsm,none}] [-o OUTPUT] [-d DIR]
[--skip_report] [-v] [-r RANDOMFOREST] [-j THRESHOLD]
sqanti_filter_classif
Rescue for ML-filtered transcriptomes.
positional arguments:
sqanti_filter_classif
SQANTI filter (ML or rules) output classification file.
optional arguments:
-h, --help show this help message and exit
--isoforms ISOFORMS FASTA file output by SQANTI3 QC (*_corrected.fasta), i.e. the full long read transcriptome.
--gtf GTF GTF file output by SQANTI3 filter (*.filtered.gtf).
-g REFGTF, --refGTF REFGTF
Full path to reference transcriptome GTF used when running SQANTI3 QC.
-f REFGENOME, --refGenome REFGENOME
Full path to reference genome FASTA used when running SQANTI3 QC.
-k REFCLASSIF, --refClassif REFCLASSIF
Full path to the classification file obtained when running SQANTI3 QC on the reference
transcriptome.
-e {all,fsm,none}, --rescue_mono_exonic {all,fsm,none}
Whether or not to include mono-exonic artifacts in the rescue. Options include: none, fsm and all
(default).
-o OUTPUT, --output OUTPUT
Prefix for output files.
-d DIR, --dir DIR Directory for output files. Default: Directory where the script was run.
--skip_report Skip creation of a report about the filtering
-v, --version Display program version number.
-r RANDOMFOREST, --randomforest RANDOMFOREST
Full path to the randomforest.RData object obtained when running the SQANTI3 ML filter.
-j THRESHOLD, --threshold THRESHOLD
Default: 0.7. Machine learning probability threshold to filter elegible rescue targets (mapping
hits).
Wiki index
- Introduction to SQANTI3
- Dependencies and installation
- Version history
- Isoform classification: categories and subcategories
- Running SQANTI3 quality control
- Understanding the output of SQANTI3 QC
- IsoAnnotLite
- Running SQANTI3 filter
- Running SQANTI3 rescue
- Tutorial: running SQANTI3 on an example dataset
- Running SQANTI-reads
- Memory requirements to use parallelization