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Running SQANTI3 rescue

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Ángeles Arzalluz-Luque edited this page Jul 1, 2022 · 33 revisions

⚠️UNDER CONSTRUCTION⚠️

Table of contents:

Introduction

As of SQANTI3 v5.1, a new module has been added to the SQANTI3 workflow for transcriptome characterization and quality control: SQANTI3 rescue.

The SQANTI3 rescue algorithm is designed to be run after transcriptome filtering and uses the long read-based evidence provided by discarded isoforms (i.e. artifacts) to recover transcripts in the associated reference transcriptome. The idea behind this strategy is to avoid losing transcripts/genes that are detected as expressed by long read sequencing, but whose start/end/junctions could not be confidently validated using orthogonal data, resulting in the removal of those genes/transcripts from the transriptome. More details about this can be found in the Motivation section below.

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

Motivation

To be completed: explain why transcript rescue is required after filtering.

Rescue strategy in SQANTI3

The SQANTI3 rescue algorithm consists in the following general steps:

1. Automatic rescue

As explained above, the rescue strategy was conceived to recover transcriptome diversity lost during filtering. This, among other things, means verifying that mone of the reference-supported junction chains that were initially detected by long-reads are lost due to stringent artifact removal.

To achieve this during automatic rescue, all reference transcripts that were represented by at least one FSM in the original post-QC transcriptome are first retrieved -note that this information is available in the associated_transcript column of the *_classification.txt file. Then, those reference transcripts for which all FSM representatives were removed by the filter are rescued.

The previous analytic decision is justified because, in practice, any case where all FSMs with the same associated_transcript are removed can be interpreted as follows: 1) the TSS and/or TTS of the long read-defined transcript is different from that of the matching reference transcript, however, it could not be validated by SQ3 QC-supplied orthogonal data; and 2) the junctions are identical to those found in the reference, which can be interpreted as evidence that this isoform is real. As a result, SQ3 will not rescue any of the discarded FSMs, but the associated_transcript from the reference.

2. Selection of rescue candidate and target transcripts

3. Mapping of candidates to targets

4. Application of SQ3 filter to reference transcriptome

5. Rescue-by-mapping

Rules filter 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.

Machine learning filter rescue

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).

Rescue output files

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