- Introduction
- Installation
- Running the MACARON
- MACARON Reporting Format
- Validating SNVs Existed on the Same Reads
- References
- Citation
MACARON (Multi-bAse Codon-Associated variant Re-annotatiON) is a python framework to identify and re-annotate multi-base affected codons in whole genome/exome sequence data. Starting from a standard VCF file, MACARON identifies, re-annotates and predicts the amino acid change resulting from multiple single nucleotide variants (SNVs) within the same genetic codon.
The information below includes how to install and run MACARON to filter a list of variant records (from VCF file) called by any existing SNP-based variant caller to identify SNVs with the same genetic codon and correct their corresponding amino acid change.
See latest News and Updates on MACARON-GenMed-LabEx Wiki page.
MACARON is know to run on LINUX UBUNTU 16.04 LTS. However, MACARON can be run on any other LINUX version.
1. MACARON is executable in PYTHON v2.7 or later. If the user has multiple PYTHON versions, please make sure that your running environment is set to the required version of PYTHON.
2. Check your JAVA version as MACARON is tested with:
java -version
openjdk version __"1.8.0_151"__
OpenJDK Runtime Environment (build 1.8.0_151-8u151-b12-0ubuntu0.16.04.2-b12)
OpenJDK 64-Bit Server VM (build 25.151-b12, mixed mode)
Before running MACARON, please make sure that following software are installed properly:
1. Genome-Analysis Toolkit (https://software.broadinstitute.org/gatk/download/).
2. SnpEff (tested with v4.3 (build 2017-05-05 18:41). However, MACARON can also run with any older or newer version (http://snpeff.sourceforge.net/download.html).
3. SAMTools (tested with version 0.1.19), however any version can be used.
4. Human Reference Genome: Depends on user’s input.
5. SnpEff’s Human Annotation Database: Depends on user’s input.
For 1 and 2, as long as they are compatible with JAVA, MACARON has no issues.
The most prefered way to use the lastest version of MACARON is:
git clone https://github.com/waqasuddinkhan/MACARON-GenMed-LabEx.git
or download the ZIP folder.
MACARON source code can also be downloaded from http://www.genmed.fr/images/publications/data/MACARON_GenMed.zip
After acquiring a release distribution of the source code, the build procedure is to unpack the zip file:
unzip MACARON_GenMed.zip
- MACARON – The MACARON python code
- MACARON_validate.sh – a BASH-shell script to validate multi-SNVs located on the same read that affect the same genetic codon
Before running MACARON, check these input technical notes as the following limitations exist for either the input VCF file, or the required software dependencines:
-
Chromosome (chr) notation should be compatible with both input VCF file and Human Reference Genome file, or vice versa,
-
Sequence dictionaries of input VCF file and Human Reference Genome file should be the same,
-
Input VCF file (should) suitably be annotated with ANNOVAR, and additionally with any other annotation software, e.g, VEP (https://www.ensembl.org/info/docs/tools/vep/index.html) if the user has a desire to get the full functionality of -f option (see Advanced Options below),
-
Same Human Reference Genome file should be used for MACARON which is practiced earlier for alignemnt and (or) to call variant sets,
-
Versions of input VCF file, Human Reference Genome file and SnpEff database file should be the same (hg19 / GRCh37 = SnpEff GRCh37.75) or (hg38 / GRCh38 = SnpEff GRCh38.86).
For a full list of MACARON executable options, run:
python MACARON -h
By default, MACARON depends on the GLOBAL VARIABLES set in the script before run:
## GLOBAL VARIABLES (IMPORTANT: You can set the default values here)
GATK="/home/wuk/software/GenomeAnalysisTK.jar"
#GATK="/home/wuk/software/gatk-4.0.1.2/gatk-package-4.0.1.2-local.jar"
HG_REF="/home/wuk/Working/gnme_refrnces/Homo_sapiens_assembly19.fasta"
SNPEFF="/home/wuk/software/snpEff/snpEff.jar"
SNPEFF_HG="GRCh37.75" ## SnpEff genome version
To run MACARON with GATK <4.0 versions, simply type:
python MACARON -i test_input.vcf
If running with GATK >= 4.0 versions, make following changes:
#GATK="/home/wuk/software/GenomeAnalysisTK.jar"
GATK="/home/wuk/software/gatk-4.0.1.2/gatk-package-4.0.1.2-local.jar"
HG_REF="/home/wuk/Working/gnme_refrnces/Homo_sapiens_assembly19.fasta"
SNPEFF="/home/wuk/software/snpEff/snpEff.jar"
SNPEFF_HG="GRCh37.75" ## SnpEff genome version
and run with:
python MACARON -i test_input.vcf --gatk4
To help verify a successful installation, MACARON includes a small demo data set:
- variants_of_interest.vcf – a test VCF file to check the functionality of MACARON
- MACARON_output.txt – The output file generated by running the MACARON
- sub1.chr22_21349676-21349677.sample02.bam – a subset of BAM file used as input for MACARON_validate.sh
- MACARON_validate.txt – The output file with read count information of concerned pcSNV in sample02 (in this case). (All files are referenced with hg19)
cd to demo folder and run:
python ../MACARON -i variants_of_interest.vcf
MACARON_output.txt is the default output file name of MACARON. User can change it with -o option.
python ../MACARON -i variants_of_interest.vcf -o variants_of_interest.txt
MACARON can be run by invoking paths directly set from the command-line:
python ../MACARON -i variants_of_interest.vcf --GATK /home/wuk/software/GenomeAnalysisTK.jar --HG_REF /home/wuk/Working/gnme_refrnces/Homo_sapiens_assembly19.fasta --SNPEFF /home/wuk/software/snpEff/snpEff.jar --SNPEFF_HG GRCh37.75- For GATK >= 4.0 versions:
python ../MACARON -i variants_of_interest.vcf --gatk4 --GATK /home/wuk/software/ --HG_REF /home/wuk/Working/gnme_refrnces/Homo_sapiens_assembly19.fasta --SNPEFF /home/wuk/software/snpEff/snpEff.jar --SNPEFF_HG GRCh37.75MACARON can add additional fields, besdies the dafault (see MACARON Reporting Format) by using -f option:
-
-f CSQ(if input VCF file is additionally annotated with VEP, the output txt file also has the same complete annotation for each variant record) -
-f EFF(if user wants to output SnpEff annotations in output txt file), or -f ANN (if SnpEff is used without -formatEff option) -
-f QUAL,DP,AF,Func.refGene,Gene.refGene,GeneDetail.refGene(this will keep any other default annotations of input VCF file and of ANNOVAR to output txt file)
-f can be used multiple times, e.g.,
-f CSQ,DP,Func.refGeneor-f FILTER,EFF,CSQ,AF
The order of the fields in the output txt file depends on the order of INFO field headers used in -f.
python ../MACARON -i variants_of_interest.vcf --gatk4 --GATK /home/wuk/software/ --HG_REF /home/wuk/Working/gnme_refrnces/Homo_sapiens_assembly19.fasta --SNPEFF /home/wuk/software/snpEff/snpEff.jar --SNPEFF_HG GRCh37.75 -f QUAL,FILTER,SIFT_pred Without -f option, QUAL field is outputted as default.If user wants to keep QUAL along with any other field, -f should mentiond QUAL in addition to other field headers: -f QUAL,FILTER,SIFT_pred. If only -f SIFT_pred is used, QUAL field is over-written by SIFT_pred field.
MACARON outputs a table text file with the following format specifications:
chr22 21349676 rs412470 T A LZTR1 423 T/T T/A T/T 0/0 0/1 0/0 MISSENSE S92T Tct Act ATt I 0 0
chr22 21349677 rs376419 C T LZTR1 423 C/C C/T C/C 0/0 0/1 0/0 MISSENSE S92F tCt tTt 0 I 0 0
| Field Number | Field Name | Description |
|---|---|---|
| 1 | CHROM | Chromosome number |
| 2 | POS | Chromosomal position / coordinates of SNV |
| 3 | ID | dbSNP rsID |
| 4 | REF | Reference base |
| 5 | ALT | Alternate base |
| 6 | Gene_Name | Name of a gene in which SnpCluster is located |
| 7 | QUAL | Quality of the ALT base called |
| 8 | [SAMPLE NAME].GT | Genotype of samples as base conventions as well as binary conventions |
| 9 | Protein_coding_EFF | Functional Effect of Variant on protein |
| 10 | AA-Change | Amino acid change by individual SNV |
| 11 | REF-codon | Reference Codon |
| 12 | ALT-codon | Alternate Codon |
| 13 | ALT-codon_merge-2VAR | A new codon formed by the combination of two Alt-codons (pcSNV codon; see MACARON) |
| 14 | AA-Change-2VAR | Re-annotated amino acid formed by pcSNV codon |
| 15 | ALT-codon_merge-3VAR | A new codon formed by the combination of three Alt-codons |
| 16 | AA-Change-3VAR | Re-annotated amino acid formed by the combination of three Alt-codons |
This default's MACARON output can be changed by using -f option. For example, if MACARON run with -f QUAL,FILTER,SIFT_pred, the new output looks like:
| Field Number | Field Name | Description |
|---|---|---|
| 1 | CHROM | Chromosome number |
| 2 | POS | Chromosomal position / coordinates of SNV |
| 3 | ID | dbSNP rsID |
| 4 | REF | Reference base |
| 5 | ALT | Alternate base |
| 6 | Gene_Name | Name of a gene in which SnpCluster is located |
| 7 | QUAL | Quality of the ALT base called |
| 8 | FILTER | Filter (PASS) tag |
| 9 | SIFT_pred | Functional effect prediction of SNV on protien |
| 10 | [SAMPLE NAME].GT | Genotype of samples as base conventions as well as binary conventions |
| 11 | Protein_coding_EFF | Functional Effect of Variant on protein |
| 12 | AA-Change | Amino acid change by individual SNV |
| 13 | REF-codon | Reference Codon |
| 14 | ALT-codon | Alternate Codon |
| 15 | ALT-codon_merge-2VAR | A new codon formed by the combination of two Alt-codons (pcSNV codon; see MACARON) |
| 16 | AA-Change-2VAR | Re-annotated amino acid formed by pcSNV codon |
| 17 | ALT-codon_merge-3VAR | A new codon formed by the combination of three Alt-codons |
| 18 | AA-Change-3VAR | Re-annotated amino acid formed by the combination of three Alt-codons |
NB: You do not need to run this step if you already used phased VCF file to run MACARON
To confirm the existence of multi-SNVs within the same genetic codon, an accessory BASH-shell script MACARON_validate.sh calculates the read count information of affected bases. This script requires as an input subset of BAM files (should be the same that used to generate the input VCF file) covering 50 bps over each SnpCluster.
Subset of any BAM file can be generated by using the following command:
samtools view –hb –L sub1.bed sample02.bam > sub1.chr22_21349676-21349677.sample02.bam
In this case, our big BAM file sample02.bam (not provided here, obviously!!!) is subsetted as sub1.chr22_21349676-21349677.sample02.bam (see demo folder) for the position chr22:21349676. The naming format of output BAM file should be the same. The sub1.bed file has 1 tab-seperated line:
chr22 21349676
representing the first position of SnpCluster (SNV1 only).
Once subset BAM file(s) are generated, run MACARON_validate.sh:
MACARON_validate.sh sub1.chr22_21349676-21349677.sample02.bam
This will generate an output text file (MACARON_validate.txt) allowing the user for further analysis.
sub1 chr22:21349676-21349677 sample02
1 AA
1 T
11 AT
14 TC
See MACARON-GenMed-LabEx Wiki page for more details, and interpretations of the demo data.
If you use MACARON in your research, please cite:
Khan W. et al. MACARON: a python framework to identify and re-annotate multi-base affected codons in whole genome/exome sequence data, Bioinformatics 2018
CONTACT: david-alexandre.tregouet@inserm.fr; waqasnayab@gmail.com, florian.thibord@gmail.com
VERSION: 1.0; VERSION RELEASE DATE: May 18, 2020