A quick and nifty tool for genotyping and phasing popular pharmacogenes.

Aldy 4 calls genotypes of many highly polymorphic pharmacogenes and reports them in a phased star-allele nomenclature. It can also call copy number of a given pharmacogene and genotype each copy present in the sample—something that standard genotype callers like GATK cannot do.

Algorithm details¶

TL;DR: Aldy 4 uses star-allele databases to guide the process of detecting the most likely genotype. The optimization is done in three stages via integer linear programming. See Gene Support for more details about the supported pharmacogene databases.

More details, together with the API documentation, are available at Read the Docs.

Experimental data is available here.

If you are using Aldy, please cite our papers in the Nature Communications and bioRxiv.

⚠️ Warning¶

Please read this carefully if you are using Aldy in a clinical or commercial environment.

Aldy is a computational tool whose purpose is to aid the genotype detection process. It can be of tremendous help in that process. However, it is not perfect, and it can easily make a wrong call if the data is noisy, ambiguous or if the target sample contains a previously unknown allele.

☣️🚨 Do not use the raw output of Aldy (or any other computational tool for that matter) to diagnose a disease or prescribe a drug! You are responsibe for inspecting and validating the results (ideally) in a wet lab before doing something that can have major consequences. 🚨☣️

We really mean it.

Finally, note that the allele databases are still a work in progress and that we still do not know the downstream impact of the vast majority of genotypes.

Installation¶

Aldy is written in Python and requires Python 3.7+ to run. It is intended to be run on POSIX-based systems (so far, only Linux and macOS have been tested).

The easiest way to install Aldy is to use pip:

pip install aldy

Append --user to the previous command to install Aldy locally if you cannot write to the system-wide Python directory.

Prerequisite: ILP solver¶

Aldy requires a mixed integer solver to run.

The following solvers are currently supported:

CBC / Google OR-Tools: a free, open-source MIP solver that is shipped by default with Google’s OR-Tools. pip installs it by default when installing Aldy.
If you have trouble installing ortools on a Nix-based Linux distro, try this:
pip install --platform=manylinux1_x86_64 --only-binary=:all: --target ~/.local/lib/python3.8/site-packages ortools
Gurobi: a commercial solver which is free for academic purposes. Most thoroughly tested solver: if you encounter any issues with CBC, try Gurobi. After installing it, don’t forget to install gurobipy package by going to Gurobi’s installation directory (e.g., /opt/gurobi/linux64 on Linux or /Library/gurobi751/mac64/ on macOS) and typing:
```
python3 setup.py install
```

Sanity check¶

After installing Aldy and a compatible ILP solver, please make sure to test the installation by issuing the following command (this should take a few minutes):

aldy test

In case everything is set up properly, you should see something like this:

🐿  Aldy v4.0 (Python 3.7.5 on macOS 12.4)
    (c) 2016-2022 Aldy Authors. All rights reserved.
    Free for non-commercial/academic use only.
================================ test session starts ================================
platform darwin -- Python 3.7.5, pytest-5.3.1, py-1.8.0, pluggy-0.13.1
rootdir: aldy, inifile: setup.cfg
plugins: anyio-3.6.1, xdist-1.31.0, cov-2.10.1, forked-1.1.3
collected 76 items
aldy/tests/test_cn_real.py ........                                            [ 10%]
aldy/tests/test_cn_synthetic.py .....                                          [ 17%]
aldy/tests/test_diplotype_real.py ....                                         [ 22%]
aldy/tests/test_diplotype_synthetic.py ......                                  [ 30%]
aldy/tests/test_full.py ...........                                            [ 44%]
aldy/tests/test_gene.py .......                                                [ 53%]
aldy/tests/test_major_real.py ...........                                      [ 68%]
aldy/tests/test_major_synthetic.py .......                                     [ 77%]
aldy/tests/test_minor_real.py .......                                          [ 86%]
aldy/tests/test_minor_synthetic.py ......                                      [ 94%]
aldy/tests/test_query.py ....                                                  [100%]
=========================== 76 passed in 131.10s (0:02:11) ==========================

Running¶

Aldy needs a SAM, BAM, CRAM or VCF file for genotyping. We will be using BAM as an example.

Attention

It is assumed that reads are mapped to hg19 (GRCh37) or hg38 (GRCh38). Other reference genomes are not yet supported.

An index is needed for BAM files. Get one by running:

samtools index file.bam

Aldy is invoked as:

aldy genotype -p [profile] -g [gene] file.bam

Sequencing profile selection¶

The [profile] argument refers to the sequencing profile. The following profiles are available:

illumina or wgs for the Illumina WGS or exome (WXS) data (or any uniform-coverage technology).

Attention

It is highly recommended to use samples with at least 40x coverage. Anything below 20x might result in noisy copy number calls and missed variants.
pgx1 for the PGRNseq v.1 capture protocol data
pgx2 for the PGRNseq v.2 capture protocol data
pgx3 for the PGRNseq v.3 capture protocol data
10x for 10X Genomics data

Attention

For the best results on the 10X Genomics datasets, use the EMA aligner, especially if doing CYP2D6 analysis. Aldy will also use the EMA read cloud information for improved variant phasing.
exome, wxs, wes for the whole-exome sequencing data

Attention

⚠️ Be warned!: whole-exome data is incomplete by definition, and Aldy will not be able to call major star-alleles defined by their intronic or upstream variants. Aldy also assumes that there are only two (2) gene copies if the wxs profile is used, as it cannot call copy number changes nor fusions from exome data.
pacbio-hifi-targeted, pacbio-hifi-targeted-twist for PacBio HiFi target capture data

Attention

The provided PacBio capture profiles are custom and are not standard. Please ensure to generate a custom profile if using different PacBio HiFi capture protocols.

If you are using a different technology (e.g., some home-brewed capture kit), you can proceed provided that the following requirements are met:

all samples have a similar coverage distribution (i.e., two sequenced samples with the same copy number configuration must have similar coverage profiles; please consult us if you are not sure about this)
your panel includes a copy-number neutral region (currently, Aldy uses CYP2D8 as a copy-number neutral region, but it can be overridden).

Having said that, you can use a sample BAM that is known to have two copies of the genes you wish to genotype (without any fusions or copy number alterations) as a profile as follows:

aldy genotype -p profile-sample.bam -g [gene] file.bam -n [cn-neutral-region]

Alternatively, you can generate a profile for your panel/technology by running:

# Get the profile
aldy profile profile-sample.bam > my-cool-tech.profile
# Run Aldy
aldy genotype -p my-cool-tech.profile -g [gene] file.bam

Note: if you are using long-read captures such as PacBio or Nanopore, make sure to add the following lines to the corresponding profile file:

options:
  sam_long_reads: true

Alternatively, you can pass this flag directly to Aldy as --param sam_long_reads=true.

Output¶

By default, Aldy will generate file-[gene].aldy (the default location can be changed via -o parameter). Aldy also supports VCF file output: to enable it, just append .vcf to the output file name. The summary of the calls is shown at the end of the output:

$ aldy -p pgx2 -g cyp2d6 NA19788.bam
🐿  Aldy v4.0 (Python 3.8.2 on Linux 3.10.0-1160.71.1.el7.x86_64-x86_64-with-glibc2.2.5)
    (c) 2016-2022 Aldy Authors. All rights reserved.
    Free for non-commercial/academic use only.
Genotyping sample NA07048.cram...
Potential CYP2D6 gene structures for NA07048:
  1: 2x*1 (confidence: 100%)
Potential major CYP2D6 star-alleles for NA07048:
  1: 1x*1, 1x*4.021 (confidence: 100%)
  2: 1x*4, 1x*139 (confidence: 100%)
  3: 1x*4.021.ALDY_2, 1x*74 (confidence: 100%)
Best CYP2D6 star-alleles for NA07048:
  1: *1 / *4.021 (confidence=100%)
      Minor alleles: *(1.016 +rs112568578 +rs113889384 +rs28371713 +rs28633410), *(4.021 +rs28371729 -rs28371702 -rs28588594)
CYP2D6 results:
  - *1 / *4.021
    Minor: [*1.016 +rs112568578 +rs113889384 +rs28371713 +rs28633410] / [*4.021 +rs28371729 -rs28371702 -rs28588594]
    Legacy notation: [*1.016 +rs112568578 +rs113889384 +rs28371713 +rs28633410] / [*4.021 +rs28371729 -rs28371702 -rs28588594]

In this example, the CYP2D6 genotype is *1/*4 in terms of major star-alleles. The minor star-alleles are given after each major star-allele call (here, *1.016 and *4.021). The minor alleles might also have additional or removed mutations. The additions are marked with + in front (e.g., +rs112568578), while the losses carry - in front (e.g., -rs28588594). In some instances, even the major alleles might contain additions (e.g., (*1 +rs1234)). This indicates the presence of a novel star-allele that has not been cataloged yet.

By default, Aldy only reports solutions with the maximum confidence. Use –param gap=XY (where XY is greater than 0) to report less likely solutions.

Explicit decomposition is given in the file-[gene].aldy (in the example above, it is NA19788_x.CYP2D6.aldy). An example of such a file is:

#Sample Gene    SolutionID      Major   Minor   Copy    Allele  Location        Type    Coverage        Effect  dbSNP   Code    Status
#Solution 1: *1.001, *4, *4.021
NA10860 CYP2D6  1       *1/*4+*4.021    1.001;4;4.021   0       1.001
NA10860 CYP2D6  1       *1/*4+*4.021    1.001;4;4.021   1       4       42522612        C>G     15      S486T   rs1135840
...[redacted]...
#Solution 2: *4, *4, *139.001
NA10860 CYP2D6  2       *4+*4/*139      4;139.001;4     0       4       42522612        C>G     15      S486T   rs1135840
NA10860 CYP2D6  2       *4+*4/*139      4;139.001;4     0       4       42524946        C>T     32      splicing defect/169frameshift    rs3892097
...[redacted]...

The columns are:

the sample name,
the gene name,
the solution count (different solutions have different counts),
the major star-allele call,
the minor star-allele call,
the allele copy identifier (0 for the first allele in the minor column, 1 for the second and so on)
the mutation location,
the mutation type (SNP or indel),
the mutation coverage,
the mutation functionality:
- DISRUPTING for gene-disrupting (functional) mutations, and
- NEUTRAL for neutral (silent) mutations
the dbSNP ID (if available),
traditional Karolinska-style mutation code from the CYP allele database (if available); and
the mutation status, which indicates the status of the mutation in the decomposition:
- NORMAL: mutation is associated with the star-allele in the database and is found in the sample
- NOVEL: gene-disrupting mutation is NOT associated with the star-allele in the database, but is found in the sample (this indicates that Aldy found a novel major star-allele)
- EXTRA: neutral mutation is NOT associated with the star-allele in the database, but is found in the sample (this indicates that Aldy found a novel minor star-allele)
- MISSING: neutral mutation is associated with the star-allele in the database, but is NOT found in the sample (this also indicates that Aldy found a novel minor star-allele)

VCF support¶

The output will be a VCF file if the output file extension is .vcf. Aldy will report a VCF sample for each potential solution and the appropriate genotypes. Aldy will also output tags MA and MI for major and minor solutions.

Note: VCF is not an optimal format for star-allele reporting. Unless you really need it, we recommend using Aldy’s default format.

Problems & Debugging¶

If you encounter any issues with Aldy, please run Aldy with debug parameter:

aldy genotype … –debug debuginfo

This will produce debuginfo.tar.gz file that contains the sample and LP model dumps. Please send us this file, and we will try to resolve the issue.

This file contains no private information of any kind except for the phasing information and mutation counts at the target gene locus as well as the file name.

Sample datasets¶

Sample datasets are also available for download. They include:

HG00463 (PGRNseq v.2), containing CYP2D6 configuration with multiple copies
NA19790 (PGRNseq v.2), containing a fusion between CYP2D6 and CYP2D7 deletion (*78 allele)
NA24027 (PGRNseq v.1), containing novel DPYD allele and multiple copies of CYP2D6
NA10856 (PGRNseq v.1), containing CYP2D6 deletion (*5 allele)
NA10860 (Illumina WGS), containing three copies of CYP2D6. This sample contains only the CYP2D6 region.

The expected results are:

Gene (-g)	HG00463	NA19790	NA24027	NA10856	NA10860
CYP2D6	36+10/36+10	1/78+*2	6/2+*2	1/5	1/4+*4
CYP2A6	1/1	1/1	1/35	1/1
CYP2C19	1/3	1/1	1/2	1/2
CYP2C8	1/1	1/3	1/3	1/1
CYP2C9	1/1	1/2	1/2	1/2
CYP3A4	1/1	1/1	1/1	1/1
CYP3A5	3/3	3/3	1/3	1/3
CYP4F2	1/1	3/4	1/1	1/1
TPMT	1/1	1/1	1/1	1/1
DPYD	1/1	1/1	4/5	5/6

License¶

Aldy is NOT free software. A complete legal license is available in aldy_license.

For non-legal folks, here is a TL;DR version:

Aldy can be freely used in academic and non-commercial environments
Please contact us if you intend to use Aldy for any commercial purpose

Parameters & Usage¶

NAME:¶

Aldy — a tool for allelic decomposition (haplotype reconstruction) and exact genotyping: of highly polymorphic and structurally variant genes.

SYNOPSIS:¶

aldy [–verbosity VERBOSITY] [–log LOG] command

Commands:

aldy help
aldy test
aldy license
aldy query (q)
aldy profile [FILE]
aldy genotype [-h] [--verbosity VERBOSITY] [--gene GENE] [--profile PROFILE]
              [--reference REFERENCE] [--genome GENOME] [--cn-neutral-region CN_NEUTRAL_REGION]
              [--output OUTPUT] [--solver SOLVER] [--debug DEBUG] [--cn CN] [--log LOG]
              [--multiple-warn-level MULTIPLE_WARN_LEVEL] [--simple]
              [--param PARAM=VALUE [PARAM2=VALUE2 ...]]
              [FILE]

OPTIONS:¶

Global arguments:¶

-h, --help

Show the help message and exit.
-v, --verbosity VERBOSITY

Logging verbosity. Acceptable values:
- T (trace)
- D (debug),
- I (info), and
- W (warn)
Default: I
-l, --log LOG

Location of the output log file.

Default: no log file

Commands:¶

help

Show the help message and exit.
license

Print Aldy license.
test

Run Aldy test suite.
query, q

Query a gene or an allele.

You can specify a gene name (e.g. aldy query CYP2D6) or an allele (e.g. aldy query 'CYP2D6*121' or aldy q 'CYP2D6*4C').
profile [FILE]

Generate a copy-number profile for a custom sequencing panel and print it on the standard output. FILE is a SAM/BAM sample that is known to have two copies of the gene of interest (without any fusions or copy number alterations).
genotype

Genotype a sequencing sample. Arguments:
- FILE
  
  A SAM, BAM, CRAM or VCF file. A CRAM file requires --reference as well.
- -p, --profile PROFILE
  
  Sequencing profile. Supported values are:
  - illumina (or wgs)
  - exome (or wxs or wes)
  - pgx1 (or pgrnseq-v1)
  - pgx2 (or pgrnseq-v2)
  - pgx3 (or pgrnseq-v3)
  - 10x
  - pacbio-hifi-targeted
  - pacbio-hifi-targeted-twist
  You can also pass a SAM/BAM file as a profile(please check the documentation quick-start for more details). Also consult profile command.
- -g, --gene GENE
  
  Gene profile.
  
  Default: CYP2D6
- -o, --output OUTPUT
  
  Location of the output file.
  
  Default: [input].[gene].aldy
- -s, --solver SOLVER
  
  ILP Solver. Currently supported solvers are Gurobi and CBC. You can also pass any to let Aldy choose the best (available) solver.
  
  Default: any (uses CBC if available, then Gurobi).
- -c, --cn CN
  
  Manually specify a copy number configuration. Input: a comma-separated list of configurations CN1,CN2,.... For a list of supported configurations, please run:
```
aldy query [GENE]
```
- -r, --reference REF
  
  FASTA reference for the reference-encoded CRAM files.
- -n, --cn-neutral-region CN_NEUTRAL
  
  Provide a custom copy-number neutral region. Format is chr:start-end.
  
  Default: CYP2D8 (22:42547463-42548249 for hg19)
- -d, --debug DEBUG
  
  Create a DEBUG.tar.gz` file that can be shared with the authors for easier debugging. Contains no private information except the file name and sample mutation counts in the gene of interest.
- --multiple-warn-level MULTIPLE_WARN_LEVEL
  
  Warning level when multiple optimal solutions are found.
  
  If set to 1, Aldy will warn if multiple final optimal solutions are found. If set to 2, Aldy will also warn if multiple optimal major star-allele solutions are found. If set to 3, Aldy will even warn if multiple copy-number configurations are found.
  
  Default: 1
- --param PARAM1=VAL1 [PARAM2=VAL2 ...]
  
  Additional model parameters. Please check the parameter documentation for the list of the available parameters.

Gene Support¶

Gene	Version	Status	Notes
CYP2D6	PharmVar 5.2.3	✅	Copy number and structural variation supported Alleles with the CYP2D7 exon 9 retention such as 36, 57, 83 and 141 can be accurately called only when the copy number detection is enabled (i.e., they cannot be called in WES mode) Detection of the non-functional CYP2D7 intron 1 retention is spotty
CYP2A6	PharmVar 5.2.3	✅	Copy number and structural variation supported Detection of the CYP2A7 3’ UTR retention not yet supported
CYP2B6	PharmVar 5.2.3	✅	Some allele calls should be further validated (e.g., 6/9)
CYP1A1	PharmGKB (Dec 2014) and Pharmacoscan R9	✅
CYP1A2	PharmGKB (Mar 2014) and Pharmacoscan R9	✅
CYP2A13	PharmVar 5.2.3	✅
CYP2C19	PharmVar 5.2.3	✅
CYP2C8	PharmVar 5.2.3	✅
CYP2C9	PharmVar 5.2.3	✅
CYP2E1	PharmGKB (Nov 2013)	⚠️	Thorough testing on the real datasets pending
CYP2F1	PharmVar 5.2.3	✅
CYP2J2	PharmVar 5.2.3	✅
CYP2R1	PharmVar 5.2.3	⚠️	Thorough testing on the real datasets pending
CYP2S1	PharmVar 5.2.3	✅
CYP2W1	PharmVar 5.2.3	⚠️	Thorough testing on the real datasets pending
CYP3A43	PharmVar 5.2.3	✅
CYP3A4	PharmVar 5.2.3	✅
CYP3A5	PharmVar 5.2.3	✅
CYP3A7	PharmVar 5.2.3	✅
CYP4F2	PharmVar 5.2.3	✅
CFTR	PharmGKB (Jun 2020) and Pharmacoscan R9	✅
COMT	Pharmacoscan R9	✅
DPYD	PharmVar 5.2.3	✅
G6PD	PharmGKB and Pharmacoscan R9 (Sep 2018)	⚠️	Thorough testing on the real datasets pending Null allele calling is unstable
GSTM1	Pharmacoscan R9	✅
GSTP1	Pharmacoscan R9	✅
IFNL3	PharmGKB and Pharmacoscan R9	✅
NAT1	PharmGKB (Mar 2014) and Pharmacoscan R9	✅
NAT2	PharmGKB (Mar 2014) and Pharmacoscan R9	✅
NUDT15	PharmVar 5.2.3	✅
SLCO1B1	PharmVar 5.2.3	✅
TPMT	PharmGKB (Jun 2020) and Pharmacoscan R9	✅
UGT1A1	PharmGKB (Feb 2020) and Pharmacoscan R9	⚠️	Thorough testing on the real datasets pending
UGT2B7	pharmacogenomics.pha.ulaval.ca (Apr 2015) / Pharmacoscan R9	⚠️	Thorough testing on the real datasets pending
VKORC1	PharmGKB (Jan 2021) and Pharmacoscan R9	⚠️	Thorough testing on the real datasets pending

Change log¶

Aldy v4.2 (Sep 25th, 2022)
- Fix indelpost setup errors
- Various small fixes
Aldy v4.1 (Aug 28th, 2022)
- Output allele’s activity and/or impact when available
- Updated and tested gene definitions - Major changes to NAT1, NAT2, UGT1A1, CYP2E1 and CYP2A6
- Indel realignment support via indelpost
- New debug format
- Various small fixes
Aldy v4.0 (Aug 17th, 2022)
- Major model changes
- Phasing support
- Long-read sequencing support (PacBio HiFi, 10X Genomics)
- Support for new pharmacogenes
- New allele databases
- New profile format (⚠️ WARNING: Please make sure to re-generate custom profiles if using older Aldy profiles.)
- Major API changes
- New debug format
- Various small fixes
Aldy v3.0 (Nov 30th, 2020)
- Support for hg38
- Support for 15+ new pharmacogenes
- New profile format (⚠️ WARNING: Please make sure to re-generate custom profiles if using Aldy v2 profiles.)
- Better genotype calling models
- Major API changes

Acknowledgments¶

The following people made Aldy much better software:

Ananth Hari
Qinghui Zhou
Michael Ford @michael-ford
Farid Rashidi @faridrashidi
David Twesigomwe @twesigomwedavid
Tyler Shrug @tshugg
Reynold C. Ly
Pieter W. Smit
Lawrence Hon @lhon
Zach Langley @zlangley

Contact & Bug Reports¶

Ibrahim Numanagić

or open a GitHub issue.

If you have an urgent problem, I suggest using e-mail.

Gene (-g)	HG00463	NA19790	NA24027	NA10856	NA10860
CYP2D6	36+10/36+10	1/78+*2	6/2+*2	1/5	1/4+*4
CYP2A6	1/1	1/1	1/35	1/1
CYP2C19	1/3	1/1	1/2	1/2
CYP2C8	1/1	1/3	1/3	1/1
CYP2C9	1/1	1/2	1/2	1/2
CYP3A4	1/1	1/1	1/1	1/1
CYP3A5	3/3	3/3	1/3	1/3
CYP4F2	1/1	3/4	1/1	1/1
TPMT	1/1	1/1	1/1	1/1
DPYD	1/1	1/1	4/5	5/6