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ABO blood typing using Oxford Nanopore MinION sequencing

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.

Required tools

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.

Testing without nextflow

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 nextflow workflow

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.sh

To run without the workload manager but with a specific containerization, use:

  • nextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -with-conda abo-analysis-env or nextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -profile conda
  • nextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -with-docker fmobegi/abo-analysis or nextflow run abo-analysis/main.nf -resume --outdir "$PWD/230128R_ABO_results" -profile docker

Results from the Nextflow pipeline will look something like this

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.txt

Feel free to raise an issue or reach out if you need any support getting this tool running, or with suggestions for improvement.