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

Aldy 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 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.

Aldy has been published in Nature Communications (doi:10.1038/s41467-018-03273-1). Preprint is available here. Full experimental pipeline is available here.

Documentation is available at Read the Docs.

Gene Support

Gene	Version	Status	Copy number & Fusion
CYP2D6	PharmVar 4.1.7	✅	✅
CYP2A6	cypalleles.ki.se (Jan 2012)	✅	✅
CYP2B6	PharmVar 4.1.7	⚠️ (possible mapping conflicts with CYP2B7; needs more validation)	🚫
CYP1A1	PharmGKB (Dec 2014)	✅	❓ (no reported CN/fusion events in the database)
CYP1A2	PharmGKB (Mar 2014)	✅	❓
CYP2A13	PharmVar 4.1.7	✅	❓
CYP2C19	PharmVar 4.1.7	✅	🚫
CYP2C8	PharmVar 4.1.7	✅	❓
CYP2C9	PharmVar 4.1.7	✅	❓
CYP2E1	PharmGKB (Nov 2013)	⚠️ (not tested on real data yet)	❓
CYP2F1	PharmVar 4.1.7	⚠️ (not tested on real data yet)	❓
CYP2J2	PharmVar 4.1.7	✅	❓
CYP2R1	PharmVar 4.1.7	⚠️ (not tested on real data yet)	❓
CYP2S1	PharmVar 4.1.7	✅	❓
CYP2W1	PharmVar 4.1.7	⚠️ (not tested on real data yet)	❓
CYP3A43	PharmVar 4.1.7	✅	❓
CYP3A4	cypalleles.ki.se (2020)	✅	❓
CYP3A5	PharmVar 4.2.4	✅	❓
CYP3A7	PharmVar 4.1.7	✅	❓
CYP4F2	PharmVar 4.1.7	✅	❓
CFTR	PharmGKB (Jun 2020)	✅	❓
COMT	Pharmacoscan	✅	❓
DPYD	PharmVar 4.1.7	✅	❓
G6PD	PharmGKB (Sep 2018)	✅	❓
GSTM1	Pharmacoscan	✅	❓
GSTP1	Pharmacoscan	✅	❓
IFNL3	PharmGKB	✅	❓
NAT1	PharmGKB (Mar 2014)	✅	❓
NAT2	PharmGKB (Mar 2014)	✅	❓
NUDT15	PharmVar 4.1.7	✅	❓
SLCO1B1	PharmGKB (Oct 2019)	✅	❓
TPMT	PharmGKB (Jun 2020)	✅	❓
UGT1A1	PharmGKB (Feb 2020)	✅	❓
UGT2B7	pharmacogenomics.pha.ulaval.ca (Apr 2015) / Pharmacoscan	✅	❓
VKORC1	PharmGKB (Jan 2021)	✅	❓

⚠️ Warning

If you are using Aldy in a clinical or commercial environment, please read this carefully.

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, ambigious 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! It is your responsibility to validate every result manually or (ideally) in a wetlab before doing something that can have major consequences. 🚨☣️

We really mean it.

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

Installation

Aldy is written in Python, and requires Python 3.7+. 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
  

• SCIP: another solver which is also free for academic purposes. SCIP is easier to install than Gurobi (no registration or activation required). However, it might be slower than Gurobi. Once you you install SCIP, please install PySCIPPpt module for the Python SCIP bindings via pip: pip install pyscipopt. If it fails, make sure to set SCIPOPTDIR environmental variable to point to SCIP's install directory.

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 v3.0 (Python 3.7.5 on macOS 10.16)
  (c) 2016-2020 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: /Users/inumanag/Projekti/aldy/devel, inifile: setup.cfg
plugins: xdist-1.31.0, forked-1.1.3
collected 73 items

tests/test_cn_real.py ........                                                                    [ 12%]
tests/test_cn_synthetic.py .....                                                                  [ 20%]
tests/test_diplotype_real.py ....                                                                 [ 27%]
tests/test_diplotype_synthetic.py ......                                                          [ 37%]
tests/test_full.py .....                                                                          [ 45%]
tests/test_gene.py ....                                                                           [ 51%]
tests/test_major_real.py ...........                                                              [ 69%]
tests/test_major_synthetic.py .......                                                             [ 80%]
tests/test_minor_real.py ......                                                                   [ 90%]
tests/test_minor_synthetic.py .....                                                               [ 98%]
tests/test_paper.py s                                                                             [100%]

=============================== 73 passed, 1 skipped in 106.83s (0:01:46) ===============================

Running

Aldy needs a SAM, BAM, or a CRAM 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 for 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 lower than 20x will result in tears and agony.

• pgx1 for PGRNseq v.1 capture protocol data

• pgx2 for PGRNseq v.2 capture protocol data

• pgx3 for PGRNseq v.3 capture protocol data

• wxs for whole-exome sequencing data

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

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

• all samples have the 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

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

Output

Aldy will by default generate the following file: file-[gene].aldy (default location can be changed via -o parameter). Aldy also supports VCF file output: just append .vcf to the output file name. The summary of results are shown at the end of the output:

$ aldy -p pgrnseq-v2 -g cyp2d6 NA19788.bam
*** Aldy v2.0 (Python 3.7.4) ***
*** (c) 2016-2019 Aldy Authors & Indiana University Bloomington. All rights reserved.
*** Free for non-commercial/academic use only.
Genotyping sample NA07048.cram...
Potential CYP2D6 copy number configurations for NA07048:
  1: 2x*1
      Confidence: 1.00 (score = 3.22)

Potential major CYP2D6 star-alleles for NA07048:
  1: 1x*1 +42525810:SNP.TC*, 1x*4.b
      Confidence: 1.00 (score = 22.47)
  2: 1x*1, 1x*4.b +42525810:SNP.TC*
      Confidence: 1.00 (score = 22.47)

Best CYP2D6 star-alleles for NA07048:
  1: *1-like/*4
      Minor: *1 +42525810:SNP.TC*, *4EW
      Confidence: 1.00 (score = 25.73)
  2: *1/*4-like
      Minor: *1, *4EW +42525810:SNP.TC*
      Confidence: 1.00 (score = 25.73)
CYP2D6 results:
  *1-like/*4                     (*1 +42525810:SNP.TC*, *4.b)
  *1/*4-like                     (*1, *4.b +42525810:SNP.TC*)

In this example, CYP2D6 genotype is *1/*4 as expressed in terms of major star-alleles. Minor star-alleles are given after each "best" star-allele (here, *1 and *4EW). Note that there is a novel SNP here (42525810:SNP.TC) that Aldy assigned to *1 (and *4 in the second solution). The presence of a novel functional SNP causes Aldy to report modified allele with the suffix -like (e.g. *1-like). Minor alleles might have additional mutations, or might lose some default mutations. Additions are marked with + in front (e.g. *1 +42525810:SNP.TC*). Losses carry - in front.

Confidence scores express Aldy's confidence in a solution. Maximum score is 1.0. By default, Aldy only reports solutions that have the confidence score of 1.0. Use --gap to report more solutions.

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

#Sample     Gene    SolutionID      Major   Minor   Copy    Allele  Location        Type    Coverage        Effect  dbSNP   Code    Status
#Solution 1: *1 +42528223:SNP.GA, *4AW, *4N -42522391:SNP.GA
NA10860     CYP2D6  1       *1/*4+*4        1;4AW;4N        0       1       42528223        SNP.GA  -1      NEUTRAL rs28588594      -1426:C>T
NA10860     CYP2D6  1       *1/*4+*4        1;4AW;4N        1       4AW     42522391        SNP.GA  -1      NEUTRAL rs28371738      4401:C>T
NA10860     CYP2D6  1       *1/*4+*4        1;4AW;4N        1       4AW     42522612        SNP.CG  -1      DISRUPTING      rs1135840       4180:G>C    ...[redacted]...
...[redacted]...
#Solution 2: *1, *4AW +42528223:SNP.GA, *4N -42522391:SNP.GA
NA10860     CYP2D6  2       *1/*4+*4        1;4AW;4N        0       1
NA10860     CYP2D6  2       *1/*4+*4        1;4AW;4N        1       4AW     42522391        SNP.GA  -1      NEUTRAL rs28371738      4401:C>T
...[redacted]...

The columns stand for: - sample name, - gene name, - solution count (different solutions have different counts), - major star-allele call, - minor star-allele call, - allele copy identifier (0 for the first allele in the minor column, 1 for the second and so on) - mutation locus, - mutation type (SNP or indel), - mutation coverage, - mutation functionality:

• DISRUPTING for gene-disrupting
• NEUTRAL for neutral mutation,

• dbSNP ID (if available),

• traditional Karolinska-style mutation code from CYP allele database, and

• 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 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 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 mutation counts at the target gene locus and 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 3 copies of CYP2D6. This sample contains only CYP2D6 region.

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

© 2016-2019 Aldy Authors, Indiana University Bloomington. All rights reserved.

Aldy is NOT free software. Complete legal license is available in :ref:`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 --- 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
aldy q
aldy profile [FILE]
aldy genotype [-h]
              --profile PROFILE
              [--verbosity VERBOSITY]
              [--gene GENE]
              [--threshold THRESHOLD]
              [--reference REFERENCE]
              [--cn-neutral-region CN_NEUTRAL_REGION]
              [--output OUTPUT]
              [--solver SOLVER]
              [--gap GAP]
              [--debug DEBUG]
              [--log LOG]
              [--fusion-penalty FUSION_PENALTY]
              [--max-minor-solutions MAX_MINOR_SOLUTIONS]
              [--multiple-warn-level MULTIPLE_WARN_LEVEL]
              [--phase PHASE]
              [--cn CN]
              [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 SAM/BAM sample. Arguments:

  • FILE

    SAM, BAM, or CRAM file. CRAM requires --reference as well.

  • -T, --threshold THRESHOLD

    Cut-off rate for variations (percent per copy). Any variation with normalized coverage less than the threshold will be ignored.

    Default: 50

  • -p, --profile PROFILE

    Sequencing profile. Supported values are:

    • illumina
    • wxs
    • pgx1
    • pgx2
    • pgx3.

    You can also pass a SAM/BAM file (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, SCIP and CBC. You can also pass any to let Aldy choose the best (available) solver.

    Default: any (uses CBC if available, then Gurobi, then SCIP).

  • -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 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)

  • -G, --gap GAP

    Solution gap. By setting this to any positive value, Aldy will also report solutions whose score is less than (1+GAP) times the optimal solution score. Useful for exploring the solution space.

    Default: 0 (only optimal solutions allowed)

  • -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.

  • -f, --fusion-penalty FUSION_PENALTY

    Penalize each fusion additional FUSION_PENALTY times. Larger values mean lower likelihood of seeing fusions.

    Default: 0.1

  • --max-minor-solutions MAX_MINOR_SOLUTIONS

    Maximum number of minor solutions to report. Default setting is to output only one even if there are multiple minor (non-functional) phases.

    Default: 1

  • --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

  • --phase PHASE

    Path to HapTree-X_<https://github.com/0xTCG/haptreex> or HapCUT2_<https://github.com/vibansal/HapCUT2> phase file that can be used to properly resolve multiple optimal solutions and generate more accurate phasing.

Change log

• 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

Acknowledgements

The following people made Aldy much better software:

• Michael Ford @michael-ford
• Farid Rashidi @faridrashidi
• David Twesigomwe @twesigomwedavid
• Tyler Shrug @tshugg
• Reynold C. Ly
• 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.