A package for filtering splice junctions extracted from noisy long read alignments generated using minimap2. These can then be used to perform second pass alignment with minimap2, feeding in the junctions using the --junc-bed flag.
2passtools has been tested with python 3.6, and requires numpy, scikit-learn, pysam, NCLS and click. The easiest way to install it is using the conda environment yaml provided:
git clone https://www.github.com/bartongroup/2passtools
cd 2passtools
conda env create -f 2passtools.yml
source activate 2passtools
Alternatively 2passtools and the required packages can be installed using pip:
pip install git+git://github.com/bartongroup/2passtools.git
2passtools has three commands....
NB: There is a snakemake pipeline which can be used to run the benchmarking scripts used in the manuscript.
The 2passtools score command requires as input a long read sequencing bam file aligned using minimap2 and a reference fasta file. It then extracts junction metrics and sequence information and uses it to score splice junctions found in the alignments. The output of score is a BED file with multiple columns corresponding to different metrics and model scores (see output below). This format cannot be passed to minimap2 directly as (A) it has not yet been filtered and (B) the extra column format is not supported by minimap2 which requires 6-column bed. Filtering and reformatting can be done using 2passtools filter.
If you already have a reference annotation but want to discover novel splice junctions, consider using the annotation-aided mode of 2passtools score. It takes an additional input: a bed file containing high-confidence splice junctions from an existing reference annotation. 2passtools will use these as positive examples to train de novo models to detect novel splice junctions. It works best if the existing annotation is relatively complete, but there are significant numbers of novel splice junctions: if the annotation is too incomplete it is better to run 2passtools using the pre-trained model, and if there are very few novel splice junctions, it is better just to do reference-guided alignment (without 2passtools). There are experiments which might help you guide your decision in the Genome Biology paper.
$ 2passtools score --help
Usage: 2passtools score [OPTIONS] BAM_FN
2passtools score: A tool for extracting and scores junctions from a bam
file aligned with minimap2. Filtered junctions can be used to realign
reads in a second pass with minimap2.
Bam file must be mapped with minimap2 and have the long form CS tag, e.g.
minimap2 -a --cs=long -k14 -x splice ref.fa reads.fq
Options:
-o, --output-bed-fn TEXT Output file path [required]
-f, --ref-fasta-fn TEXT Path to the fasta file that reads were
mapped to [required]
-a, --annot-bed-fn TEXT Optional BED file containing annotated
junctions
-j, --jad-size-threshold INTEGER
JAD to threshold at in the decision tree
-d, --primary-splice-local-dist INTEGER
Distance to search for alternative
donor/acceptors when calculating primary d/a
-m, --canonical-motifs TEXT Intron motifs considered canonical in
organism. Should be four char DNA motifs
separated by vertical bar only
-w, --lr-window-size INTEGER Sequence size to extract to train logistic
regression models
-k, --lr-kfold INTEGER Number of cross validation k-folds for
logistic regression models
-lt, --lr-low-confidence-threshold FLOAT
Logistic regression low confidence threshold
for decision tree 2
-ht, --lr-high-confidence-threshold FLOAT
Logistic regression high confidence
threshold for decision tree 2
-c, --classifier-type [decision_tree|random_forest]
When annotated juncs are available, train
this classifier type
--keep-all-annot / --filter-annot
When annotated juncs are available, always
keep all annotated juncs
--stranded / --unstranded Whether input data is stranded or
unstranded. direct RNA is stranded, cDNA
often isn't
-p, --processes INTEGER
-s, --random-seed INTEGER
-v, --verbosity LVL Either CRITICAL, ERROR, WARNING, INFO or
DEBUG
--help Show this message and exit.
A 13-column BED file format with the following values:
1. chrom (string)
2. start (integer)
3. end (integer)
4. intron-motif (four char string)
5. supporting read count (integer)
6. strand (string, either '+' or '-')
7. junction alignment distance metric (integer)
8. primary donor metric (integer, either 0 or 1)
9. primary acceptor metric (integer, either 0 or 1)
10. decision tree 1 output (integer, either 0 or 1)
11. logistic regression model donor score (float)
12. logistic regression model acceptor score (float)
13. decision tree 2 output (integer, either 0 or 1)
The 2passtools filter command can be used to filter the 13-column bed file using any expression utilising the metrics or model outputs. The expression should be a valid python expression which evaluates to True or False for each junction, and can use any of the following safe variables and functions:
motif: The intron motif in ACGTN alphabet (str),is_GTAG: The intron motif is GU/AG (bool),is_GCAG: The intron motif is GC/AG (bool),is_ATAG: The intron motif is AU/AG (bool),motif_regex_match: safe function allowing regex matching of motif, e.g.motif_regex_match("G[CT]AG")(func),count: The supporting read count (int),jad: The junction alignment distance metric (int),primary_donor: The primary donor metric (bool),primary_acceptor: The primary acceptor metric (bool),donor_seq_score: The logistic regression model donor score (float),acceptor_seq_score: The logistic regression model acceptor score (float),decision_tree_1_pred: Decision tree model 1 output (bool),decision_tree_2_pred: Decision tree model 2 output (bool),sum,pow,min,max,len: python functions,math: The pythonmathmodule, any function from it is useable,bool,int,str,float: python functions.
For example:
2passtools filter --exprs 'jad > 3'filters for junction alignment distance of 4 nt or more.2passtools filter --exprs 'decision_tree_2_pred'filters for junctions that pass the second decision tree model.
etc.
$ 2passtools filter --help
Usage: 2passtools filter [OPTIONS] BED_FN
2passtools filter: Convenience tool to filter a junction bed and produce
6-column bed format which is compatible with minimap2.
Options:
-o, --output-bed-fn TEXT [required]
--exprs TEXT
-v, --verbosity LVL Either CRITICAL, ERROR, WARNING, INFO or DEBUG
--help Show this message and exit.
The 2passtools merge command is similar to score, but takes multiple 13-column bed files produced by score and merges them, recalculating metrics and model stats, to produce a unified junction set. This is useful for making sure all replicates are aligned similarly, and often alignment is improved by borrowing power across replicates. Output is in the same 13-column BED format as score.
$ 2passtools merge --help
Usage: 2passtools merge [OPTIONS] BED_FNS...
2passtools merge: Merges bed files produced by 2passtools score on
individual replicates and recalculates junction strength metrics. Can be
used to create a unified junction set to realign reads from different
replicates.
Bed files should be in the 13 column format produced by 2passtools score.
Options:
-o, --output-bed-fn TEXT Output file path [required]
-f, --ref-fasta-fn TEXT Path to the fasta file that reads were
mapped to [required]
-a, --annot-bed-fn TEXT Optional BED file containing annotated
junctions
-j, --jad-size-threshold INTEGER
JAD to threshold at in the decision tree
-d, --primary-splice-local-dist INTEGER
Distance to search for alternative
donor/acceptors when calculating primary d/a
-m, --canonical-motifs TEXT Intron motifs considered canonical in
organism. Should be four char DNA motifs
separated by vertical bar only
-w, --lr-window-size INTEGER Sequence size to extract to train logistic
regression models
-k, --lr-kfold INTEGER Number of cross validation k-folds for
logistic regression models
-lt, --lr-low-confidence-threshold FLOAT
Logistic regression low confidence threshold
for decision tree 2
-ht, --lr-high-confidence-threshold FLOAT
Logistic regression high confidence
threshold for decision tree 2
-c, --classifier-type [decision_tree|random_forest]
When annotated juncs are available, train
this classifier type
--keep-all-annot / --filter-annot
When annotated juncs are available, always
keep all annotated juncs
-p, --processes INTEGER
-s, --random-seed INTEGER
-v, --verbosity LVL Either CRITICAL, ERROR, WARNING, INFO or
DEBUG
--help Show this message and exit.
The 2passtools manuscript is published in Genome Biology (Open access):
Parker, M.T., Knop, K., Barton, G.J. et al. 2passtools: two-pass alignment using machine-learning-filtered splice junctions increases the accuracy of intron detection in long-read RNA sequencing. Genome Biol 22, 72 (2021). https://doi.org/10.1186/s13059-021-02296-0