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FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing.

ABSTRACT: Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

SUBMITTER: Kinde I

PROVIDER: S-EPMC3399813 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing.

Kinde Isaac I Papadopoulos Nickolas N Kinzler Kenneth W KW Vogelstein Bert B

PloS one 20120718 7

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% triso ...[more]

PMID: 22815955

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Project description:BackgroundCirculating 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.MethodsWhole 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.ResultsTwo 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.ConclusionsThese data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Dataset Information

FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing.

Publications

FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing.

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