RHtyper

Predicts RHD/RHCE genotypes using whole-genome sequencing (WGS) data

RHtyper - prediction of RH alleles

Please read this important information before running the app

Introduction

The RHD and RHCE genes encode Rh blood group antigens and exhibit extensive nucleotide polymorphisms and chromosome structural changes, often resulting in expression of Rh variant antigens. RH variants are common in people of African descent including patients of sickle cell disease and can drive loss of antigen epitopes or expression of new antigen epitopes, predisposing these patients to Rh alloimmunization. Serologic antigen typing is limited to common Rh antigens, necessitating a genetic approach to detect variant antigen expression. RHtyper is a novel algorithm developed for comprehensive RH genotyping from WGS data.

Installation

RHtyper with WES platform support can be installed from the Conda. NOTE: We suggest to use Mamba to speed up the installation process (https://mamba.readthedocs.io/en/latest/mamba-installation.html#mamba-install). After mamba installed, RHtyper can be installed with following steps:

1. mamba create -n RHtyper
2. mamba install -c ticchang rhtyper

What does RHtyper do?

RHtyper predicts RHD and RHCE genotypes by using short-read WGS data, specifically WGS alignment files in BAM (*.bam) format generated by short read aligners.

How does RHtyper work?

RHtyper predicts RHD and RHCE genotypes through the following main steps:

1. Variant calling;
2. Determining RHD gene zygosity and allele zygosity by coverage profiles and variant information; and
3. Likelihood scoring and ranking to predict the most fit RH allele pairs.

What data parameters does RHtyper require?

1. Required data and data parameters:
   • Short-read WGS alignment files (.bam)
   • Index of the BAM alignment files (.bai)
   • Version of human reference genome
   • Output prefix (added to output files)
   • Estimated coverage depth of the WGS data
2. Optional data and adjustable data parameters:
   • Customized reference genome sequence; users can provide a customized reference genome sequence that is used for generating the alignment files
   • Customized allele linkage database; users can provide customized RH allele linkage information specific to the study population (If not specified, RHtyper uses haplotype association information from the International Society of Blood Transfusion (ISBT) database)
   • Customized allele population frequency database; users can provide a customized allele population frequency database specific to the study population (If not specified, RHtyper uses a default database that is updated continually)
   • Gene(s) for genotyping; users can select RHD and RHCE, RHD only, or RHCE only
   • Cutoff of alternative nucleotide read number for variant calling

What data format is required for the allele linkage and allele population frequency databases?

Both databases are in tab-delimited text format and can be created using Excel (with headers). Examples are shown below.

1. Allele linkage database:

Gene	Allele name	Allele detail	Alias	Linked	comment
RHCE	RHCE*01.02.01 RHCE*ce.02.01	RHCE*ce48C,1025T	RHCE*ceTI	RHD*04.01_RHD*DIVa
RHCE	RHCE*01.05.01 RHCE*ce.05.01	RHCE*ce48C,712G,787G,800A	RHCE*ceEK	RHD*DAR
RHD	RHD*04.01 RHD*DIVa	RHD*186T,410T,455C,1048C		RHCE*01.02.01 RHCE*ce.02.01
...	...	...	...	...	...

2. Allele population frequency database:

Allele name	Nucleotide	Allele detail	Alias	PopFreq
RHD*10.00 RHD*DAU0	c.1136C>T	RHD*1136T	RHD*1136T	0.1651
RHD*09.03.01 RHD*DAR3.01	c.602C>G; c.667T>G; c.819G>A	RHD*602G,667G	RHD*602G,667G	0.0298
RHCE*02 or RHCE*Ce RHCE*C RHCE*e	c.48G>C		RHCE*Ce	0.119
...	...	...	...	...

What RHtyper outputs are included?

1. Main output:
   • bloodtyping.pdf - Predicted alleles, variants, and coverage profiles in a PDF file
   • bloodtyping.xlsx - Information of typed allele pairs and ranking of allele pairs in an Excel worksheet
2. Other text outputs that can be used for aggregated analyses:
   • bloodtyping.txt - Predicted genotypes in a cohort of samples in a tab-delimited text format
   • exonCNV.txt - Copy number variation status per exon in a tab-delimited text format
   • final.variant.txt - Variant identified in a tab-delimited text format

How to run RHtyper?

NOTE: the latest DNAnexus web interface is used for the following workflow

1. Create a "New Project". If you have an existing project, please skip to step 5.

2. Name the New Project, select “Azure US (West),” and click “Create Project”

3. Upload BAM and BAI files: Under MANAGE, click "Add", and select "Upload Data"

4. Upload BAM and BAI files following the instructions on the screen

5. Find RHtyper: Click the search icon and type “RHtyper”

6. Select "RHtyper"

7. "Install" RHtyper for the first-time user. Note: A readme and tutorial of RHtyper can be found on this page.

8. Click "Run" button to set up a new analysis

9. Select the project created at step1 or other projects containing the alignment BAM files for analyses.

10. Select a folder for output results: Under Analysis Settings, click "Execution Output Folder"

11. Create an output folder: Create a "New Folder" or select an exiting folder, then click "Select Folder"

12. Provide input data information: Under Analysis Inputs, select "BAM" and "BAI" files and fill the required information (*) about your input data.

13. Adjust output settings under "Stage Settings" section. Click "Run Analysis" button to start the RHtyping. NOTE: the instance type can be adjusted to increase memory for a run.

14. Output is saved in the folder specified at step10.