ABO sequences were aquired from the NCBI dbRBC database:
https://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.cgi?cmd=bgmut/home
See https://ftp.ncbi.nlm.nih.gov/pub/mhc/rbc/Final%20Archive/Excel_and_PowerPoint/ for some literature.
The pipeline makes use of the following core dependencies:
- bioconda::fastqc=0.12.1
- bioconda::bwa=0.7.17
- conda-forge::ncurses
- bioconda::samtools=1.19.2
- bioconda::minimap2=2.26
- conda-forge::biopython=1.83
- python=3.10
- pip
- pip:
- numpy>=1.26.0
- Bio>=1.6.0
- biopython>=1.8o
- openpyxl>=3.1.0
- pandas>=2.2.0
- pysam>=0.22.0
- matplotlib>=3.8.0
- XlsxWriter>=3.2.0
- multiqc>=1.18
A complete list of dependencies is found in the assets folder assets/conda.yml.
The pipeline can be tested of single input file by cloning this repo and installing all dependncies above, then running the following commands:
python bin/AnalyzeAbo_Main.py \
--reference="assets/A1_01_01_1_reference_Exon6.fasta" \
--alleles="assets/ABO_Database.fasta" \
--output="SampleName/exon6" \
--analysis-type="READS" \
--reads="SampleName.fastq" \
python bin/AnalyzeAbo_Main.py \
--reference="assets/reads_bc51/A1_01_01_1_reference_Exon7.fasta" \
--alleles="assets/input/ABO_Database.fasta" \
--output="SampleName/exon7" \
--analysis-type="READS" \
--reads="SampleName.fastq"Looping through a couple of samples with the above command will generate the following outputs:
Data structure
OutputDirectoryName/
├── Sample1
│ ├── exon6
│ │ └── alignment
│ └── exon7
│ └── alignment
├── Sample2
│ ├── exon6
│ │ └── alignment
│ └── exon7
│ └── alignment
├── Sample3
│ ├── exon6
│ │ └── alignment
│ └── exon7
│ └── alignment
With individual files named as follows:
OutputDirectoryName/
├── Sample1
│ ├── exon6
│ │ ├── ABOPhenotype.txt
│ │ ├── ABOReadPolymorphisms.txt
│ │ ├── alignment
│ │ │ ├── alignment.bam
│ │ │ ├── alignment.bam.bai
│ │ │ ├── AlignmentReference.fasta
│ │ │ ├── AlignmentReference.fasta.amb
│ │ │ ├── AlignmentReference.fasta.ann
│ │ │ ├── AlignmentReference.fasta.bwt
│ │ │ ├── AlignmentReference.fasta.pac
│ │ │ └── AlignmentReference.fasta.sa
│ │ └── ReadAlignmentSpreadsheet.csv
│ ├── exon7
│ │ ├── ABOPhenotype.txt
│ │ ├── ABOReadPolymorphisms.txt
│ │ ├── alignment
│ │ │ ├── alignment.bam
│ │ │ ├── alignment.bam.bai
│ │ │ ├── AlignmentReference.fasta
│ │ │ ├── AlignmentReference.fasta.amb
│ │ │ ├── AlignmentReference.fasta.ann
│ │ │ ├── AlignmentReference.fasta.bwt
│ │ │ ├── AlignmentReference.fasta.pac
│ │ │ └── AlignmentReference.fasta.sa
│ │ └── ReadAlignmentSpreadsheet.csv
│ ├── Sample1_exon6.log.txt
│ └── Sample1_exon7.log.txt
The ABOPhenotype.txt files from each sampe can then be collated using:
python bin/Aggregate_ABO_reports.py OutputDirectoryName
The steps above are simplified in a NextFlow; https://www.nextflow.io/ pipeline that does all the above steps and streamlines installation of requisite software and tools with a single command.
Besides reproducability, nextflow offeres several advatages over conventional for loops, including scallability, portability, and debugging/resumption of failed tasks.
Input files and output directory can be defined in the config files or provided directly in the commandline.
To analyse files with config, run:
nextflow run main.nf -resume(user can override inputs and output using--reads '*.fastq' --outdir 'ABO_results'on the commandline).
We have also added the ability for the pipeline to automatically set-up a conda or docker based environment with all required tools and libraries.
Users may also opt for a workload manager such as -profile slurm,docker|-profile slurm,conda, is which case, all required modules docker/conda must be installed and loaded. The config slurm parameters must also be defined to ensure tasks are submitted to the correct resource queue/account.
For conda environment, it is advisable to prepare the working computer using mamba for easy resolution of environments.
Follow these steps to achieve better results.
mamba create -y -n abo-analysis-env
conda activate abo-analysis-env
mamba env update --file abo-analysis/assets/conda.yml --prune
conda deactivate
# If conda cativate fails, run:
source {path_to_anaconda}/anaconda3/etc/profile.d/conda.shTo run without the workload manager but with a specific containerization, use:
nextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -with-conda abo-analysis-envornextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -profile condanextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -with-docker fmobegi/abo-analysisornextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -profile docker
230128R_ABO_results/
├── ABO_result.txt
├── ABO_result.xlsx
├── execution_report.html
├── execution_timeline.html
├── execution_trace.txt
├── SampleName
│ ├── exon6
│ │ ├── ABOPhenotype.txt
│ │ ├── ABOReadPolymorphisms.txt
│ │ ├── alignment
│ │ │ ├── alignment.bam
│ │ │ ├── alignment.bam.bai
│ │ │ ├── AlignmentReference.fasta
│ │ │ ├── AlignmentReference.fasta.amb
│ │ │ ├── AlignmentReference.fasta.ann
│ │ │ ├── AlignmentReference.fasta.bwt
│ │ │ ├── AlignmentReference.fasta.pac
│ │ │ └── AlignmentReference.fasta.sa
│ │ └── ReadAlignmentSpreadsheet.csv
│ ├── exon7
│ │ ├── ABOPhenotype.txt
│ │ ├── ABOReadPolymorphisms.txt
│ │ ├── alignment
│ │ │ ├── alignment.bam
│ │ │ ├── alignment.bam.bai
│ │ │ ├── AlignmentReference.fasta
│ │ │ ├── AlignmentReference.fasta.amb
│ │ │ ├── AlignmentReference.fasta.ann
│ │ │ ├── AlignmentReference.fasta.bwt
│ │ │ ├── AlignmentReference.fasta.pac
│ │ │ └── AlignmentReference.fasta.sa
│ │ └── ReadAlignmentSpreadsheet.csv
│ ├── SampleName_exon6.log.txt
│ └── SampleName_exon7.log.txt
├── software_versions.txt
└── workflow.oncomplete.txtFeel free to raise an issue or reach out if you need any support getting this tool running, or with suggestions for improvement.