Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection

Marija Debeljak, Donald N. Freed, Jane A. Welch, Lisa Haley, Katie Beierl, Brian S. Iglehart, Aparna Pallavajjalla, Christopher Gocke, Mary S. Leffell, Ming-Tseh Lin, Jonathan A. Pevsner, Sarah Wheelan, James Eshleman

Research output: Contribution to journalArticle

Abstract

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.

Original languageEnglish (US)
Pages (from-to)495-503
Number of pages9
JournalJournal of Molecular Diagnostics
Volume16
Issue number5
DOIs
StatePublished - 2014

Fingerprint

Haplotypes
Single Nucleotide Polymorphism
Hematopoietic Stem Cell Transplantation
Microsatellite Repeats
HLA-A Antigens
Bone Marrow
DNA
Tissue Donors
Paternity
Chimerism
DNA Primers
Capillary Electrophoresis
Organ Transplantation
Human Genome
Limit of Detection
Alleles
Genome
Databases
Recurrence
Cell Line

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection. / Debeljak, Marija; Freed, Donald N.; Welch, Jane A.; Haley, Lisa; Beierl, Katie; Iglehart, Brian S.; Pallavajjalla, Aparna; Gocke, Christopher; Leffell, Mary S.; Lin, Ming-Tseh; Pevsner, Jonathan A.; Wheelan, Sarah; Eshleman, James.

In: Journal of Molecular Diagnostics, Vol. 16, No. 5, 2014, p. 495-503.

Research output: Contribution to journalArticle

Debeljak, Marija ; Freed, Donald N. ; Welch, Jane A. ; Haley, Lisa ; Beierl, Katie ; Iglehart, Brian S. ; Pallavajjalla, Aparna ; Gocke, Christopher ; Leffell, Mary S. ; Lin, Ming-Tseh ; Pevsner, Jonathan A. ; Wheelan, Sarah ; Eshleman, James. / Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection. In: Journal of Molecular Diagnostics. 2014 ; Vol. 16, No. 5. pp. 495-503.
@article{440cc66150e345edb06b9e75f60ee823,
title = "Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection",
abstract = "Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01{\%}. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0{\%} to 1.5{\%} patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.",
author = "Marija Debeljak and Freed, {Donald N.} and Welch, {Jane A.} and Lisa Haley and Katie Beierl and Iglehart, {Brian S.} and Aparna Pallavajjalla and Christopher Gocke and Leffell, {Mary S.} and Ming-Tseh Lin and Pevsner, {Jonathan A.} and Sarah Wheelan and James Eshleman",
year = "2014",
doi = "10.1016/j.jmoldx.2014.04.003",
language = "English (US)",
volume = "16",
pages = "495--503",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Association of Molecular Pathology",
number = "5",

}

TY - JOUR

T1 - Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection

AU - Debeljak, Marija

AU - Freed, Donald N.

AU - Welch, Jane A.

AU - Haley, Lisa

AU - Beierl, Katie

AU - Iglehart, Brian S.

AU - Pallavajjalla, Aparna

AU - Gocke, Christopher

AU - Leffell, Mary S.

AU - Lin, Ming-Tseh

AU - Pevsner, Jonathan A.

AU - Wheelan, Sarah

AU - Eshleman, James

PY - 2014

Y1 - 2014

N2 - Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.

AB - Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.

UR - http://www.scopus.com/inward/record.url?scp=84906248336&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84906248336&partnerID=8YFLogxK

U2 - 10.1016/j.jmoldx.2014.04.003

DO - 10.1016/j.jmoldx.2014.04.003

M3 - Article

C2 - 25132481

AN - SCOPUS:84906248336

VL - 16

SP - 495

EP - 503

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

IS - 5

ER -