Using Targeted Sequencing of Paralogous Sequences for Noninvasive Detection of Selected Fetal Aneuploidies.
BACKGROUND
Current methods for noninvasive prenatal testing (NIPT) ascertain fetal aneuploidies using either direct counting measures of DNA fragments from specific genomic regions or relative measures of single nucleotide polymorphism frequencies. Alternatively, the ratios of paralogous sequence pairs were predicted to reflect fetal aneuploidy. We developed a NIPT assay that uses paralog sequences to enable noninvasive detection of fetal trisomy 21 (T21) and trisomy 18 (T18) using cell-free DNA (cfDNA) from maternal plasma.


METHODS
A total of 1060 primer pairs were designed to determine fetal aneuploidy status, fetal sex, and fetal fraction. Each library was prepared from cfDNA by coamplifying all 1060 target pairs together in a single reaction well. Products were measured using massively parallel sequencing and deviations from expected paralog ratios were determined based on the read depth from each paralog.


RESULTS
We evaluated this assay in a blinded set of 480 cfDNA samples with fetal aneuploidy status determined by the MaterniT21®PLUS assay. Samples were sequenced (mean = 2.3 million reads) with 432 samples returning a result. Using the MaterniT21 PLUS assay for paired plasma aliquots from the same individuals as a reference, all 385 euploid samples, all 31 T21 samples, and 14 of 16 T18 samples were detected with no false positive results observed.


CONCLUSIONS
This study introduces a novel NIPT aneuploidy detection approach using targeted sequencing of paralog motifs and establishes proof-of-concept for a potentially low-cost, highly scalable method for the identification of selected fetal aneuploidies with performance and nonreportable rate similar to other published methods.
Sequenom Laboratories and.
Christopher K
CK
Ellison
Sequenom Laboratories and.
Youting
Y
Sun
Sequenom Laboratories and.
Grant
G
Hogg
Sequenom Laboratories and.
Jesse
J
Fox
Sequenom Laboratories and.
Helen
H
Tao
Sequenom Laboratories and.
Erin
E
McCarthy
Sequenom Laboratories and.
Bright
B
Sagoe
Sequenom Laboratories and.
Mostafa A
MA
Azab
Sequenom Laboratories and.
Amin R
AR
Mazloom
Sequenom Laboratories and.
John
J
Tynan
Sequenom, Inc., San Diego, California.
Timothy
T
Burcham
Sequenom Laboratories and.
Sung K
SK
Kim
Sequenom, Inc., San Diego, California.
Dirk
D
van den Boom
Sequenom, Inc., San Diego, California.
Mathias
M
Ehrich
Sequenom Laboratories and tjensen@sequenom.com.
Taylor J
TJ
Jensen
D000782:Aneuploidy; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D011296:Prenatal Diagnosis; D017422:Sequence Analysis, DNA
D004247:DNA