IsoQuant 3.3 manual

1. About IsoQuant
   1.1. Supported data types
   1.2. Supported reference data
   
2. Installation
   2.1. Installing from conda
   2.2. Manual installation and requirements
   2.3. Verifying your installation
   
3. Running IsoQuant
   3.1. IsoQuant input
   3.2. Command line options
   3.3. IsoQuant output
   
4. Citation
   
5. Feedback and bug reports
   

Quick start:

• IsoQuant can be downloaded from https://github.com/ablab/IsoQuant or installed via conda:

  conda create -c conda-forge -c bioconda -n isoquant python=3.8 isoquant
  

• If installing manually, you will need Python3 (3.8 or higher), gffutils, pysam, pybedtools, biopython and some other common Python libraries to be installed. See requirements.txt for details. You will also need to have minimap2 and samtools to be in your $PATH variable.

• Verify your installation by running:

  isoquant.py --test
  

• To run IsoQuant on raw FASTQ/FASTA files use the following command

  isoquant.py --reference /PATH/TO/reference_genome.fasta \
  --genedb /PATH/TO/gene_annotation.gtf \
  --fastq /PATH/TO/sample1.fastq.gz /PATH/TO/sample2.fastq.gz \
  --data_type (assembly|pacbio_ccs|nanopore) -o OUTPUT_FOLDER
  

  For example, using the toy data provided within this repository,

  ./isoquant.py --reference tests/toy_data/MAPT.Mouse.reference.fasta \
  --genedb tests/toy_data/MAPT.Mouse.genedb.gtf \
  --fastq tests/toy_data/MAPT.Mouse.ONT.simulated.fastq \
  --data_type nanopore -o toy_data_out
  

• To run IsoQuant on aligned reads (make sure your BAM is sorted and indexed) use the following command:

    isoquant.py --reference /PATH/TO/reference_genome.fasta \
    --genedb /PATH/TO/gene_annotation.gtf \
    --bam /PATH/TO/sample1.sorted.bam /PATH/TO/sample2.sorted.bam \
    --data_type (assembly|pacbio_ccs|nanopore) -o OUTPUT_FOLDER
  

  For example, using the toy data provided within this repository,

    ./isoquant.py --reference tests/toy_data/MAPT.Mouse.reference.fasta \
    --genedb tests/toy_data/MAPT.Mouse.genedb.gtf \
    --fastq tests/toy_data/MAPT.Mouse.ONT.simulated.fastq \
    --data_type nanopore -o toy_data_out
  

• If using official annotations containing gene and transcript features use --complete_genedb to save time.

• Using reference annotation is optional since version 3.0, you may preform de novo transcript discovery without providing --genedb option':

    isoquant.py --reference /PATH/TO/reference_genome.fasta \
    --fastq /PATH/TO/sample1.fastq.gz /PATH/TO/sample2.fastq.gz \
    --data_type (assembly|pacbio|nanopore) -o OUTPUT_FOLDER
  

• If multiple files are provided, IsoQuant will create a single output annotation and a single set of gene/transcript expression tables.

About IsoQuant

IsoQuant is a tool for the genome-based analysis of long RNA reads, such as PacBio or Oxford Nanopores. IsoQuant allows to reconstruct and quantify transcript models with high precision and decent recall. If the reference annotation is given, IsoQuant also assigns reads to the annotated isoforms based on their intron and exon structure. IsoQuant further performs annotated gene, isoform, exon and intron quantification. If reads are grouped (e.g. according to cell type), counts are reported according to the provided grouping.

IsoQuant consists of two stages, which generate its own output:

1. Reference-based analysis. Runs only if reference annotation is provided. Performs read-to-isofrom assignment, splice site correction and abundance quantification for reference genes/transcripts.
2. Transcript discovery. Reconstructs transcript models and performs abundance quantification for discovered isoforms.

IsoQuant version 3.3.1 was released under GPLv2 on July 26th, 2023 and can be downloaded from https://github.com/ablab/IsoQuant.

IsoQuant pipeline

Supported data types

IsoQuant support all kinds of long RNA data:

• PacBio CCS
• ONT dRNA / ONT cDNA
• Assembled / corrected transcript sequences

Reads must be provided in FASTQ or FASTA format (can be gzipped). If you have already aligned your reads to the reference genome, simply provide sorted and indexed BAM files.

Supported reference data

Reference genome should be provided in multi-FASTA format (can be gzipped). Reference genome is mandatory even when BAM files are provided.

Reference gene annotation is not mandatory, but is likely to increase precision and recall. It can be provided in GFF/GTF format (can be gzipped). In this case it will be converted to gffutils database. Information on converted databases will be stored in your ~/.config/IsoQuant/db_config.json to increase speed of future runs. You can also provide gffutils database manually. Make sure that chromosome/scaffold names are identical in FASTA file and gene annotation.

Pre-constructed aligner index can also be provided to increase mapping time.

Installation

IsoQuant requires a 64-bit Linux system or Mac OS and Python (3.8 and higher) to be pre-installed on it. You will also need

• gffutils
• pysam
• biopython
• pybedtools
• pyfaidx
• pandas
• pyyaml
• minimap2
• samtools
• STAR (optional)

Installing from conda

IsoQuant can be installed with conda:

conda install -c bioconda isoquant

Manual installation and requirements

To obtain IsoQuant you can download repository and install requirements.
Clone IsoQuant repository and switch to the latest release:

git clone https://github.com/ablab/IsoQuant.git
cd IsoQuant
git checkout latest

Install requirements:

pip install -r requirements.txt

You also need samtools and minimap2 to be in the $PATH variable.

Verifying your installation

To verify IsoQuant installation type

isoquant.py --test

to run on toy dataset.
If the installation is successful, you will find the following information at the end of the log:

=== IsoQuant pipeline finished ===
=== TEST PASSED CORRECTLY ===

Running IsoQuant

IsoQuant input

To run IsoQuant, you should provide:

• Long RNA reads (PacBio or Oxford Nanopore) in one of the following formats:
  • FASTA/FASTQ (can be gzipped);
  • Sorted and indexed BAM;
• Reference sequence in FASTA format (can be gzipped);
• Optionally, you may provide a reference gene annotation in gffutils database or GTF/GFF format (can be gzipped).

IsoQuant is also capable of using short Illumina reads to correct long-read alignments.

IsoQuant can handle data from multiple experiments simultaneously. Each experiment may contain multiple samples (or replicas). Each experiment is processed individually. Running IsoQuant on several experiments simultaneously is equivalent to several separate IsoQuant runs.

The output files for each experiment will be placed into a separate folder. Files from the same experiment are used to construct a single GTF and aggregated abundance tables. If a single experiment contains multiple samples/replicas, per sample abundance tables are also generated.

The ways of providing input files are described below.

Specifying input data via command line

Two main options are --fastq and --bam (see description below). Both options accept one or multiple files separated by space. All provided files are treated as a single experiment, which means a single combined GTF will be generated. If multiple files are provided, IsoQuant will compute tables with each column corresponding to an individual file (per-sample counts). To set a specific label for each sample use the --label option. Number of labels must be equal to the number of files. To a set a prefix for the output files use the --prefix option.

This pipeline is typical for the cases when a user is interested in comparing expression between different replicas/conditions within the same experiment.

Short reads for alignment correction

A BAM file with Illumina reads can be provided via --illumina_bam. It cannot be the only input, but may only be used with either --bam or --fastq. The option accepts one or multiple bam files separated by space. All files will be combined and used to correct offsets between introns in long and short reads as well as skipped exons.

Specifying input data via yaml file

To provide all input files in a single description file, you can use a YAML file via --yaml (see description below). You can provide multiple experiments in a single YAML file with each experiment containing an arbitrary number of smaples/replicas. A distinct output folder with individual GTFs and abundance tables will be generated for each experiment. In this option, BAM files with short reads for correction can be provided for each experiment.

The YAML file contains a list of experiments (e.g. in square brackets). The first entry in the list should be the type of files the experiments contain, written as data format: followed by the type in quotation marks. The type can be either fastq or bam.

Each experiment is represented as set of parameters (e.g. in curly brackets). Each experiment must have a name and a list if long-read files in the specified format. Additionally, it may contain one or multiple BAM files with short reads. The name is provided as name: followed by the experiment name in quotation marks. Both short and long read files are provided as a list of file paths in quotation marks, following long read files: and illumina bam: respectively. Labels for the files can also be set with labels: . The number of labels needs to be the same as the number of files with long reads. All paths should be either absolute or relative to the YAML file.

For example:

[
  data format: "fastq",
  {
    name: "Experiment1",
    long read files: [
      "/PATH/TO/FILE1.fastq",
      "/PATH/TO/FILE2.fastq"
    ],
    labels: [
      "Sample1",
      "Sample2"
    ],
    illumina bam: ["PATH/TO/ILLUMINA1.bam"]
  },
  {
    name: "Experiment2",
    long read files: [
      "/PATH/TO/FILE3.fastq"
    ],
    illumina bam: ["PATH/TO/ILLUMINA2.bam"]
  }
]

Output sub-folders will be named Experiment1 and Experiment2. Both sub-folders will contain predicted transcript models and abundance tables. Abundance table for Experiment2 with have columns "Sample1" and "Sample2".

Note, that --bam, --fastq and --label options are not compatible with --yaml. See more in examples.

Specifying input data via dataset description file (deprecated)

This option is deprecated since version 3.4 and will be removed later. To process multiple experiments, please use --yaml instead.

If you wish to process several independent experiments in a single run, you should provide a dataset description file via --fastq_list or --bam_list (see description below). A distinct output folder with individual GTFs and abundance tables will be generated for each experiment.

Input files should be provided one per line. Experiments should be separated by blank lines or experiment names starting with #. You can also set a specific label for each listed file using colon. For example:

#EXPERIMENT1
/PATH/TO/FILE1A.fastq:SAMPLE_A
/PATH/TO/FILE2A.fastq:SAMPLE_A
/PATH/TO/FILE1B.fastq:SAMPLE_B
/PATH/TO/FILE2B.fastq:SAMPLE_B
#EXPERIMENT2
/PATH/TO/FILE3.fastq:SAMPLE_C1
/PATH/TO/FILE4.fastq:SAMPLE_C2

Output sub-folders will be named EXPERIMENT1 and EXPERIMENT2. Abundance tables will have specified labels as column names. If you want to group multiple files as a single sample within the experiment, use identical labels.

Note, that --label option has no effect in this case. See more in examples.

IsoQuant command line options

Basic options

--output (or -o) Output folder, will be created automatically.

Note: if your output folder is located on a shared disk, use --genedb_output for storing reference annotation database.

--help (or -h) Prints help message.

--full_help Prints all available options (including hidden ones).

--test Runs IsoQuant on the toy data set.

Input options

--data_type or -d Type of data to process, supported values are: pacbio_ccs (same as pacbio), nanopore (same as ont) and assembly (same as transcripts). This option affects the algorithm parameters.

Note, that for novel mono-exonic transcripts are not reported for ONT data by default, use --report_novel_unspliced true.

--reference or -r Reference genome in FASTA format (can be gzipped), required even when BAM files are provided.

--index Reference genome index for the specified aligner (minimap2 by default), can be provided only when raw reads are used as an input (constructed automatically if not set).

--genedb or -g Gene database in gffutils database format or GTF/GFF format (can be gzipped). If you use official gene annotations we recommend to set --complete_genedb option.

--complete_genedb Set this flag if gene annotation contains transcript and gene meta-features. Use this flag when providing official annotations, e.g. GENCODE. This option will set disable_infer_transcripts and disable_infer_genes gffutils options, which dramatically speeds up gene database conversion (see more here).

Providing input reads via command line option:

--fastq Input FASTQ/FASTA file(s), can be gzipped; a single GTF will be generated for all files. If multiple files are provided, expression tables with "per-file" columns will be computed. See more about input data.

--bam Sorted and indexed BAM file(s); a single GTF will be generated for all files. If multiple files are provided, expression tables with "per-file" columns will be computed. See more about input data.

Providing input reads via YAML configuration file:

--yaml Path to dataset description file in YAML format. The file should contain a list with data format property, which can be fastq or bam and an individual entry for experiment. Each experiment is represented as set of parameters (e.g. in curly brackets):

• name - experiment name, string (optional);
• long read files - a list of paths to long read files matching the specified format;
• lables - a list labels for long read files for expression table (optional, must be equal to the number of long read files)
• illumina bam - a list of paths to short read BAM files for splice site correction (optional).

All paths should be either absolute or relative to the YAML file. See more in examples.

Providing input reads via dataset description file (deprecated)

--bam_list Text file with list of BAM files, one file per line. Each file must be sorted and indexed. Leave empty line or experiment name starting with # between the experiments. For each experiment IsoQuant will generate a individual GTF and count tables. You may also give a label for each file specifying it after a colon (e.g. /PATH/TO/file.bam:replicate1).

--fastq_list Text file with list of FASTQ/FASTA files (can be gzipped), one file per line. Leave empty line or experiment name starting with # between the experiments. For each experiment IsoQuant will generate a individual GTF and count tables. You may also give a label for each file specifying it after a colon (e.g. /PATH/TO/file.fastq:replicate1).

Other input options:

--stranded Reads strandness type, supported values are: forward, reverse, none.

--fl_data Input sequences represent full-length transcripts; both ends of the sequence are considered to be reliable.

--prefix or -p Prefix for all output files and sub-folder name. OUT if not set.

--labels or -l Sets space-separated sample names. Make sure that the number of labels is equal to the number of files. Input file names are used as labels if not set.

--read_group Sets a way to group feature counts (e.g. by cell type). Available options are:

• file_name: groups reads by their original file names (or file name labels) within an experiment. This option makes sense when multiple files are provided. This option is designed for obtaining expression tables with a separate column for each file. If multiple BAM/FASTQ files are provided and --read_group option is not set, IsoQuant will set --read_group:file_name by default.
• tag: groups reads by BAM file read tag: set tag:TAG, where TAG is the desired tag name (e.g. tag:RG with use RG values as groups, RG will be used if unset);
• read_id: groups reads by read name suffix: set read_id:DELIM where DELIM is the symbol/string by which the read id will be split (e.g. if DELIM is _, for read m54158_180727_042959_59310706_ccs_NEU the group will set as NEU);
• file: uses additional file with group information for every read: file:FILE:READ_COL:GROUP_COL:DELIM, where FILE is the file name, READ_COL is column with read ids (0 if not set), GROUP_COL is column with group ids (1 if not set), DELIM is separator symbol (tab if not set). File can be gzipped.

Output options

--sqanti_output Produce comparison between novel and known transcripts in SQANTI-like format. Will take effect only when reference annotation is provided.

--check_canonical Report whether read or constructed transcript model contains non-canonical splice junction (requires more time).

--count_exons Perform exon and intron counting in addition to gene and transcript counting. Will take effect only when reference annotation is provided.

Pipeline options

--resume Resume a previously unfinished run. Output folder with previous run must be specified. Allowed options are --threads and --debug, other options cannot be changed. IsoQuant will run from the beginning if the output folder does not contain the previous run.

--force force to overwrite the folder with previous run.

--threads or -t Number of threads to use, 16 by default.

--clean_start Do not use previously generated gene database, genome indices or BAM files, run pipeline from the very beginning (will take more time).

--no_model_construction Do not report transcript models, run read assignment and quantification of reference features only.

--run_aligner_only Align reads to the reference without running IsoQuant itself.

Algorithm parameters

Quantification

--transcript_quantification Transcript quantification strategy, should be one of:

• unique_only - only reads that are uniquely assigned and consistent with a transcript are used for quantification;
• with_ambiguous - ambiguously assigned reads are split between transcripts with equal weights (e.g. 1/2 when a read is assigned to 2 transcripts simultaneously, default mode);
• with_inconsistent - uniquely assigned reads with non-intronic inconsistencies (i.e. alternative poly-A site, TSS etc) are also included;
• all - all of the above.

--gene_quantification Gene quantification strategy, should be one of:

• unique_only - only reads that are uniquely assigned to a gene and consistent with any of gene's isoforms are used for quantification;
• with_ambiguous - ambiguously assigned reads are split between genes with equal weights (e.g. 1/2 when a read is assigned to 2 genes simultaneously);
• with_inconsistent - only reads that are uniquely assigned to a gene but not necessarily consistent with genes isoforms (default);
• all - all of the above.

Read to isoform matching:

--matching_strategy A preset of parameters for read-to-isoform matching algorithm, should be one of:

• exact - delta = 0, all minor errors are treated as inconsistencies;
• precise - delta = 4, only minor alignment errors are allowed, default for PacBio data;
• default - delta = 6, alignment errors typical for Nanopore reads are allowed, short novel introns are treated as deletions;
• loose - delta = 12, even more serious inconsistencies are ignored, ambiguity is resolved based on nucleotide similarity.

Matching strategy is chosen automatically based on specified data type. However, the parameters will be overridden if the matching strategy is set manually.

Read alignment correction:

--splice_correction_strategy A preset of parameters for read alignment correction algorithms, should be one of:

• none - no correction is applied;
• default_pacbio - optimal settings for PacBio CCS reads;
• default_ont - optimal settings for ONT reads;
• conservative_ont - conservative settings for ONT reads, only incorrect splice junction and skipped exons are fixed;
• assembly - optimal settings for a transcriptome assembly;
• all - correct all discovered minor inconsistencies, may result in overcorrection.

This option is chosen automatically based on specified data type, but will be overridden if set manually.

Transcript model construction:

--model_construction_strategy A preset of parameters for transcript model construction algorithm, should be one of

• reliable - only the most abundant and reliable transcripts are reported, precise, but not sensitive;
• default_pacbio - optimal settings for PacBio CCS reads;
• sensitive_pacbio - sensitive settings for PacBio CCS reads, more transcripts are reported possibly at a cost of precision;
• fl_pacbio - optimal settings for full-length PacBio CCS reads, will be used if --data_type pacbio_ccs and --fl_data options are set;
• default_ont - optimal settings for ONT reads, novel mono-exonic transcripts are not reported (use --report_novel_unspliced true);
• sensitive_ont - sensitive settings for ONT reads, more transcripts are reported possibly at a cost of precision (including novel mono-exonic isoforms);
• assembly - optimal settings for a transcriptome assembly: input sequences are considered to be reliable and each transcript to be represented only once, so abundance is not considered;
• all - reports almost all novel transcripts, loses precision in favor to recall.

This option is chosen automatically based on specified data type, but will be overridden if set manually.

--report_novel_unspliced Report novel mono-exonic transcripts (set true or false). The default value is false for Nanopore data and true for other data types. The main explanation that some aligner report a lot of false unspliced alignments for ONT reads.

Hidden options

Options below are shown only with --full_help option. We recommend not to modify these options unless you are clearly aware of their effect.

--no_secondary Ignore secondary alignments.

--report_unstranded Report transcripts for which the strand cannot be detected using canonical splice sites.

--aligner Force to use this alignment method, can be starlong or minimap2; minimap2 is currently used as default. Make sure the specified aligner is in the $PATH variable.

--no_junc_bed Do not use annotation for read mapping.

--junc_bed_file Annotation in BED12 format produced by paftools.js gff2bed (can be found in minimap2), will be created automatically if not given.

--delta Delta for inexact splice junction comparison, chosen automatically based on data type.

--genedb_output If your output folder is located on a shared storage (e.g. NFS share), use this option to set another path for storing the annotation database, because SQLite database cannot be created on a shared disks. The folder will be created automatically.

--low_memory Deprecated, default behaviour since 3.2.

--high_memory Cache read alignments instead for making several passes over a BAM file, noticeably increases RAM usage.

--min_mapq Filers out all alignments with MAPQ less than this value (will also filter all secondary alignments, as they typically have MAPQ = 0).

--multi_intron_mapping_quality_cutoff Filers out inconsistent alignments with MAPQ less than this value (works when the reference annotation is provided, default is 5).

--simple_alignments_mapq_cutoff Filers out alignments with 1 or 2 exons and MAPQ less than this value (works only in annotation-free mode, default is 1).

Examples

• Mapped PacBio CCS reads in BAM format; pre-converted gene annotation:

isoquant.py -d pacbio_ccs --bam mapped_reads.bam \
 --genedb annotation.db --output output_dir

• Nanopore dRNA stranded reads; official annotation in GTF format, use custon prefix for output:

isoquant.py -d nanopore --stranded forward --fastq ONT.raw.fastq.gz \
 --reference reference.fasta --genedb annotation.gtf --complete_genedb \
 --output output_dir --prefix My_ONT

• Nanopore cDNA reads; no reference annotation:

isoquant.py -d nanopore --fastq ONT.cDNA.raw.fastq.gz \
 --reference reference.fasta --output output_dir --prefix My_ONT_cDNA

• PacBio FL reads; custom annotation in GTF format, which contains only exon features:

isoquant.py -d pacbio_ccs --fl_data --fastq CCS.fastq \
 --reference reference.fasta --genedb genes.gtf --output output_dir

• Nanopore cDNA reads, multiple samples/replicas within a single experiment; official annotation in GTF format:

isoquant.py -d nanopore --bam ONT.cDNA_1.bam ONT.cDNA_2.bam ONT.cDNA_3.bam \
 --reference reference.fasta --genedb annotation.gtf --complete_genedb --output output_dir
 --predix ONT_3samples --labels A1 A2 A3

• ONT cDNA reads; 2 experiments with 3 replicates; official annotation in GTF format:

isoquant.py -d nanopore --yaml dataset.yaml  \
 --complete_genedb --genedb genes.gtf \
 --reference reference.fasta --output output_dir

dataset.yaml file :

[
  data format: "fastq",
  {
    name: "Experiment1",
    long read files: [
      "/PATH/TO/SAMPLE1/file1.fastq",
      "/PATH/TO/SAMPLE1/file2.fastq",
      "/PATH/TO/SAMPLE1/file3.fastq"
    ],
    labels: [
      "Replicate1",
      "Replicate2",
      "Replicate3"
    ]
  },
  {
    name: "Experiment1",
    long read files: [
      "/PATH/TO/SAMPLE2/file1.fastq",
      "/PATH/TO/SAMPLE2/file2.fastq",
      "/PATH/TO/SAMPLE2/file3.fastq"
    ],
    labels: [
      "Replicate1",
      "Replicate2",
      "Replicate3"
    ]
  }
]

IsoQuant will produce 2 sets of resulting files (including annotations and expression tables), one for each experiment. Output sub-folder will be named Experiment1 and Experiment2. Expression tables will have columns "Replicate1", "Replicate2" and "Replicate3".

• ONT cDNA reads; 1 experiment with 2 replicates, each replicate has 2 files; official annotation in GTF format:

isoquant.py -d nanopore --yaml dataset.yaml  \
  --complete_genedb --genedb genes.gtf \
 --reference reference.fasta --prefix MY_SAMPLE \
 --output output_dir  

dataset.yaml file :

[
  data format: "fastq",
  {
    name: "Experiment1",
    long read files: [
      "/PATH/TO/SAMPLE1/file1.fastq",
      "/PATH/TO/SAMPLE1/file2.fastq",
      "/PATH/TO/SAMPLE1/file3.fastq",
      "/PATH/TO/SAMPLE1/file3.fastq"
    ],
    labels: [
      "Replicate1",
      "Replicate1",
      "Replicate2",
      "Replicate2"
    ]
  }
]

IsoQuant will produce one output sub-folder Experiment1. Expression tables will have columns "Replicate1" and "Replicate2". Files having identical labels will be treated as a single replica (and thus the counts will be combined).

IsoQuant output

Output files

IsoQuant output files will be stored in <output_dir>, which is set by the user. If the output directory was not specified the files are stored in isoquant_output.

IsoQuant consists of two stages, which generate its own output:

1. Reference-based analysis. Runs only if reference annotation is provided. Performs read-to-isofrom assignment, splice site correction and abundance quantification for reference genes/transcripts.
2. Transcript discovery. Reconstructs transcript models and performs abundance quantification for discovered isoforms.

Reference-based analysis output

• SAMPLE_ID.read_assignments.tsv - TSV file with read to isoform assignments;
• SAMPLE_ID.corrected_reads.bed - BED file with corrected read alignments;
• SAMPLE_ID.transcript_tpm.tsv - TSV file with reference transcript expression in TPM;
• SAMPLE_ID.transcript_counts.tsv - TSV file with raw read counts for reference transcript;
• SAMPLE_ID.gene_tpm.tsv - TSV file with reference gene expression in TPM;
• SAMPLE_ID.gene_counts.tsv - TSV file with raw read counts for reference genes;

If --sqanti_output is set, IsoQuant will produce output in SQANTI-like format:

• SAMPLE_ID.novel_vs_known.SQANTI-like.tsv - discovered novel transcripts vs reference transcripts (similar, but not identical to SQANTI classification.txt);

If --count_exons is set, exon and intron counts will be produced:

• SAMPLE_ID.exon_counts.tsv - reference exon inclusion/exclusion read counts;
• SAMPLE_ID.intron_counts.tsv - reference intron inclusion/exclusion read counts;

If --read_group is set, the per-group expression values for reference features will be also computed:

• SAMPLE_ID.gene_grouped_tpm.tsv
• SAMPLE_ID.transcript_grouped_tpm.tsv
• SAMPLE_ID.gene_grouped_counts.tsv
• SAMPLE_ID.transcript_grouped_counts.tsv
• SAMPLE_ID.exon_grouped_counts.tsv
• SAMPLE_ID.intron_grouped_counts.tsv

Transcript discovery output

File names typically contain transcript_model in their name.

• SAMPLE_ID.transcript_models.gtf - GTF file with discovered expressed transcript (both known and novel transcripts);
• SAMPLE_ID.transcript_model_reads.tsv - TSV file indicating which reads contributed to transcript models;
• SAMPLE_ID.transcript_model_tpm.tsv - expression of discovered transcripts models in TPM (corresponds to SAMPLE_ID.transcript_models.gtf);
• SAMPLE_ID.transcript_model_counts.tsv - raw read counts for discovered transcript models (corresponds to SAMPLE_ID.transcript_models.gtf);
• SAMPLE_ID.extended_annotation.gtf - GTF file with the entire reference annotation plus all discovered novel transcripts;

If --read_group is set, the per-group counts for discovered transcripts will be also computed:

• SAMPLE_ID.transcript_model_grouped_counts.tsv
• SAMPLE_ID.transcript_model_grouped_tpm.tsv

If multiple experiments are provided, aggregated expression matrices will be placed in <output_dir>:

• combined_gene_counts.tsv
• combined_gene_tpm.tsv
• combined_transcript_counts.tsv
• combined_transcript_tpm.tsv

Additionally, a log file will be saved to the directory.

• <output_dir>/isoquant.log

If raw reads were provided, BAM file(s) will be stored in <output_dir>/<SAMPLE_ID>/aux/.
In case --keep_tmp option was specified this directory will also contain temporary files.

Output file formats

Although most output files include headers that describe the data, a brief explanation of the output files is provided below.

Read to isoform assignment

Tab-separated values, the columns are:

• read_id - read id;
• chr - chromosome id;
• strand - strand of the assigned isoform (not to be confused with read mapping strand);
• isoform_id - isoform id to which the read was assigned;
• gene_id - gene id to which the read was assigned;
• assignment_type - assignment type, can be:
  • unique - reads was unambiguously assigned to a single known isoform;
  • unique_minor_difference - read was assigned uniquely but has alignment artifacts;
  • inconsistent - read was matched with inconsistencies, closest match(es) are reported;
  • ambiguous - read was assigned to multiple isoforms equally well;
  • noninfomative - reads is intronic/intergenic.
• assignment_events - list of detected inconsistencies; for each assigned isoform a list of detected inconsistencies relative to the respective isoform is stored; values in each list are separated by + symbol, lists are separated by comma, the number of lists equals to the number of assigned isoforms; possible events are (see graphical representation below):
  • consistent events:
    • none / . / undefined - no special event detected;
    • mono_exon_match mono-exonic read matched to mono-exonic transcript;
    • fsm - full splice match;
    • ism_5/3 - incomplete splice match, truncated on 5'/3' side;
    • ism_internal - incomplete splice match, truncated on both sides;
    • mono_exonic - mono-exonic read matching spliced isoform;
    • tss_match / tss_match_precise - 5' read is located less than 50 / delta bases from the TSS of the assigned isoform
    • tes_match / tes_match_precise - 3' read is located less than 50 / delta bases from the TES of the assigned isoform (can be reported without detecting polyA sites)
  • alignment artifacts:
    • intron_shift - intron that seems to be shifted due to misalignment (typical for Nanopores);
    • exon_misalignment - short exon that seems to be missed due to misalignment (typical for Nanopores);
    • fake_terminal_exon_5/3 - short terminal exon at 5'/3' end that looks like an alignment artifact (typical for Nanopores);
    • terminal_exon_misalignment_5/3 - missed reference short terminal exon;
    • exon_elongation_5/3 - minor exon extension at 5'/3' end (not exceeding 30bp);
    • fake_micro_intron_retention - short annotated introns are often missed by the aligners and thus are not considered as intron retention;
  • intron retentions:
    • intron_retention - intron retention;
    • unspliced_intron_retention - intron retention by mono-exonic read;
    • incomplete_intron_retention_5/3 - terminal exon at 5'/3' end partially covers adjacent intron;
  • significant inconsistencies (each type end with _known if all resulting read introns are annotated and _novel otherwise):
    • major_exon_elongation_5/3 - significant exon extension at 5'/3' end (exceeding 30bp);
    • extra_intron_5/3 - additional intron on the 5'/3' end of the isoform;
    • extra_intron - read contains additional intron in the middle of exon;
    • alt_donor_site - read contains alternative donor site;
    • alt_acceptor_site - read contains alternative annotated acceptor site;
    • intron_migration - read contains alternative annotated intron of approximately the same length as in the isoform;
    • intron_alternation - read contains alternative intron, which doesn't fall intro any of the categories above;
    • mutually_exclusive_exons - read contains different exon(s) of the same total length comparing to the isoform;
    • exon_skipping - read skips exon(s) comparing to the isoform;
    • exon_merge - read skips exon(s) comparing to the isoform, but a sequence of a similar length is attached to a neighboring exon;
    • exon_gain - read contains additional exon(s) comparing to the isoform;
    • exon_detach - read contains additional exon(s) comparing to the isoform, but a neighboring exon looses a sequnce of a similar length;
    • terminal_exon_shift - read has alternative terminal exon;
    • alternative_structure - reads has different intron chain that does not fall into any of categories above;
  • alternative transcription start / end (reported when poly-A tails are present):
    • alternative_polya_site - read has alternative polyadenylation site;
    • internal_polya_site - poly-A tail detected but seems to be originated from A-rich intronic region;
    • correct_polya_site - poly-A site matches reference transcript end;
    • aligned_polya_tail - poly-A tail aligns to the reference;
    • alternative_tss - alternative transcription start site.
• exons - list of coordinates for normalized read exons (1-based, indels and polyA exons are excluded);
• additional - field for supplementary information, which may include:
  • PolyA - True if poly-A tail is detected;
  • Canonical - True if all read introns are canonical, Unspliced is used for mono-exon reads; (use --check_canonical)

Note, that a single read may occur more than once if assigned ambiguously.

Expression table format

Tab-separated values, the columns are:

• feature_id - genomic feature ID;
• TPM or count - expression value (float).

For grouped counts, each column contains expression values of a respective group. In the number of groups exceeds 10, file will contain 3 columns:

• feature_id - genomic feature ID;
• group_id - name of the assigned group;
• TPM or count - expression value (float).

Exon and intron count format

Tab-separated values, the columns are:

• chr - chromosome ID;
• start - feature leftmost 1-based positions;
• end - feature rightmost 1-based positions;
• strand - feature strand;
• flags - symbolic feature flags, can contain the following characters:
  • X - terminal feature;
  • I - internal feature;
  • T - feature appears as both terminal and internal in different isoforms;
  • S - feature has similar positions to some other feature;
  • C - feature is contained in another feature;
  • U - unique feature, appears only in a single known isoform;
  • M - feature appears in multiple different genes.
• gene_ids - list if gene ids feature belong to;
• group_id - read group if provided (NA by default);
• include_counts - number of reads that include this feature;
• exclude_counts - number of reads that span, but do not include this feature;

Transcript models format

Constructed transcript models are stored in usual GTF format. Contains exon, transcript and gene features. Transcript ids have the following format: transcript_###.TYPE, where ### is the unique number (not necessarily consecutive) and TYPE can be one of the following:

• known - previously annotated transcripts;
• nic - novel in catalog, new transcript that contains only annotated introns;
• nnic - novel not in catalog, new transcript that contains unannotated introns.

The attribute field also contains gene_id (either matches reference gene id or can be novel_gene_###), reference_gene_id (same value) and reference_transcript_id (either original isoform id or novel). In addition, it contains canonical property if --check_canonical is set.

Event classification figures

Consistent match classifications

Misalignment classifications

Inconsistency classifications

PolyA classifications

Citation

The paper describing IsoQuant algorithms and benchmarking is available at 10.1038/s41587-022-01565-y.

To try IsoQuant you can use the data that was used in the publication zenodo.org/record/7611877.

Feedback and bug reports

Your comments, bug reports, and suggestions are very welcome. They will help us to further improve IsoQuant. If you have any troubles running IsoQuant, please send us isoquant.log from the <output_dir> directory.

You can leave your comments and bug reports at our GitHub repository tracker or send them via email: [email protected].