Creating a Targeted Next Generation Sequencing Assay to Identify Rh antigens

from DNA for Stem Cell Transplants

Abigail Joseph

Mentors: William Lane MD, PhD; Peter Tonellato PhD; Helen Mah, MS

August 10th, 2016

Molecular Typing vs. Serologic Blood Typing

• RBCA (red blood cell antigens) are typed serologically.

• HLA (human leukocyte antigens) are typed molecularly.

• There are no FDA approved molecular typing tests for ABO and RhD

Stem Cell Transplants

• Treatment of Leukemia or Lymphoma may require HLA matched stem cell transplants.

• Only DNA is available during unrelated donor screening.

• ABO and RhD testing require blood samples.

• Often patients receive ABO and RhD incompatible transplants.

RHD and RHCE Genes

Question: Can we create a targeted NGS (next generation sequencing) assay that can specifically sequence RHD and RHCE ?

Methods

Long Range PCR with

Primer Sets

1

Tagmentation and Indexing

DNA fragmentation and RHD and RHCE DNA barcodes

2

Illumina Miseq

DNA NGS

3

Burrows Wheeler

Alignment Algorithm

Alignment of reads to reference genome

4

RHD and RHCE Long Range PCR Gelsst

anda

rdst

anda

rd

stan

dard

stan

dard

Sequence Alignments

Exon 1 Exon 2 Exon 3

Ref Gene Seq

Alignment and Primer Results

• The BWA (Burrows Wheeler Alignment Algorithm) aligns the DNA reads. Coverage varies significantly based on the primers used in the PCR.

RHD

Exon 1

Different primer sets used

RHCE

Exon 3

Phenotype Prediction

• However, when one is C+, exon 2 of RHCE and some of the surrounding intron are identical to the same part of RHD, so it aligns there instead.

RHCE RHD

• The sample only contained RHCE DNA.

Exon 2Exon 3 Exon 2

Phenotype Prediction

• The base at position c.676 p.pro228 in exon 5 of RHCE determines whether one has the EE, Ee, or ee genotype

c.676 p.pro228

Exon 5

Conclusion

• We tested multiple primer sets to determine which provided the most successful alignments.

• We determined the patient’s genotype to be D+, C+, c-, E, e+.

• This assay could be easily adopted in typing stem cell donors.

One DNA sample could provide a donor's entire blood type

Acknowledgements

Dana-Farber/Harvard Cancer Center

Brigham and Women’s Hospital Lab of Pathology

Continuing Umbrella of Research Experiences (CURE) ProgramKaren Burns WhiteEmily McMains

MentorsDr. Peter Tonellato Dr. William LaneHelen Mah

ColleaguesJohn BaronasSaul Granados

FunderA. David Mazzone Awards Program