1. 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

2. 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

3. 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.

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

5. 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

6. RHD and RHCE Long Range PCR Gels
standardstandard
standardstandard

7. Sequence Alignments
Exon 1 Exon 2 Exon 3
Ref
Gene
Seq

8. 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

9. 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

10. 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

11. 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

12. Acknowledgements
Dana-Farber/Harvard Cancer Center
Brigham and Women’s Hospital Lab of Pathology
Continuing Umbrella of Research Experiences (CURE) Program
Karen Burns White
Emily McMains
Mentors
Dr. Peter Tonellato
Dr. William Lane
Helen Mah
Colleagues
John Baronas
Saul Granados
Funder
A. David Mazzone Awards Program