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