ABO blood group typing using third-generation sequencing (TGS) technology

Introduction

The ABO system classifies human blood into exclusive types of O, A, B and AB based on the presence or absence of specific A and B antigens on the surface of red blood cells. Traditional phenotyping of ABO blood groups using serological methods has transformed recipient and donor profiling for blood transfusion, solid organ, or stem cell transplantation. However, in registry typing by buccal swabs, or samples of cord blood where erythrocytes are unavailable, or when serological testing is inconclusive or not possible due to the lack of antisera, molecular genotyping is essential. Molecular blood group typing using Nanopore MinION sequencing (provided by Oxford Nanopore Technologies) has aided in the characterization of polymorphisms responsible for most of the clinically relevant blood group antigens

Code structure

The base code is split into 7 Python scripts.
• Aggregate_ABO_reports.py
• AnalyzeAbo.py
• AnalyzeAbo_Common.py
• AnalyzeAbo_Main.py
• BloodGroupStats.py
• generate_samples_mapping.py
• PairwiseAlignmentResult.py
The Python scripts utilized in this workflow were adapted from the original code made available under the Creative Commons (CC) license by the transplant immunology department of Maastricht University Medical Centre (https://github.com/transplantation-immunology-maastricht/abo-analysis).

Dependencies

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Purpose	Tool	Version
Reads QC	FastQC	0.12.1
Local alignment of FASTQ to reference	BWA Minimap2 (update)	0.7.17-r1188 2.26-r1175
Processing BAM files	SAMtools	1.19.2
Quantifying variants	Python + pip packages    Bio    biopython    openpyxl    pandas    pysam    xlsxwriter    scipy	3.10    1.6.2    1.83    3..1.9    2.2.0    0.22.0    3.1.9    1.12.0
Reporting	MultiQC	1.18

Using reference GRCh38.p14 (hg38), polymorphisms on exon 6 at bp 261 (Chr9:133257521; rs8176719), exon 7 at bp 796 (Chr9:133255935; rs8176746), and exon 7 at bp 803 (Chr9:133255928; rs8176747) were used. Specific combinations and proportions of these polymorphisms can be used to distinguish between the ABO blood group types as summarised below.

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Phenotype	Genotype	Exon 6 (bp 261)	Exon 7 (bp 796)	Exon 7 (bp 803)
O	OO	Del 100%	C 100%	G 100%
A	AA	G 100%	C 100%	G 100%
B	BB	G 100%	A 100%	C 100%
AB	AB	G 100%	C 50%/ A 50%	G 50%/ C 50%
B	OB	Del 50%/ G 50%	C 50%/ A 50%	G 50%/ C 50%
A	OA	Del 50%/ G 50%	C 100%	G 100%