title: High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.
abstract: BACKGROUND
  Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.
  
  
  METHODS
  Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.
  
  
  RESULTS
  Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.
  
  
  CONCLUSIONS
  These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.
authors.affiliation: Research and Development, Sequenom Center for Molecular Medicine, San Diego, CA, USA.
authors.forename: Taylor J
authors.initials: TJ
authors.lastname: Jensen
authors.forename: Tricia
authors.initials: T
authors.lastname: Zwiefelhofer
authors.forename: Roger C
authors.initials: RC
authors.lastname: Tim
authors.forename: Željko
authors.initials: Ž
authors.lastname: Džakula
authors.forename: Sung K
authors.initials: SK
authors.lastname: Kim
authors.forename: Amin R
authors.initials: AR
authors.lastname: Mazloom
authors.forename: Zhanyang
authors.initials: Z
authors.lastname: Zhu
authors.forename: John
authors.initials: J
authors.lastname: Tynan
authors.forename: Tim
authors.initials: T
authors.lastname: Lu
authors.forename: Graham
authors.initials: G
authors.lastname: McLennan
authors.forename: Glenn E
authors.initials: GE
authors.lastname: Palomaki
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Canick
authors.forename: Paul
authors.initials: P
authors.lastname: Oeth
authors.forename: Cosmin
authors.initials: C
authors.lastname: Deciu
authors.forename: Dirk
authors.initials: D
authors.lastname: van den Boom
authors.forename: Mathias
authors.initials: M
authors.lastname: Ehrich
mesh_terms: D000782:Aneuploidy; D004247:DNA / Q000097:blood*; D005260:Female; D005333:Fetus / Q000473:pathology*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D011247:Pregnancy; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA