Accurate Fetal Chromosome Dosage Determination by Shotgun Sequencing of Maternal Plasma DNA without PCR Amplification during Library Preparation

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To the Editor: Nextgen sequencing technologies have enabled noninvasive prenatal testing of fetal aneuploidies by shotgun sequencing of maternal plasma DNA during pregnancy (1). A statistically significant change (e.g., using z -score statistics) in the number of reads mapped to a chromosome of interest (such as chromosome 21) is used to identify fetal aneuploidy. The accuracy of the test is largely dependent on 2 factors: the variability (SD) of the percentage of reads mapped to the chromosome of interest among euploidy samples and the absolute difference between the sample being tested and the mean of the euploidy samples. We speculated that the PCR enrichment step in the library preparation before cluster generation and sequencing contributes to the variation in chromosome dosage determination. Additionally, with 3 ng of DNA (typically obtainable from about 2 mL of maternal plasma) at an average size of 150 bp per DNA fragment, around 20 billion DNA fragments were available for sequencing, suggesting PCR amplification may not be necessary. We thus modified the current protocol for library preparation from maternal plasma DNA by removing the PCR enrichment step. Maternal plasma samples of about 2.5–3.2 mL were obtained with informed consent. DNA preparation and sequencing library preparation were performed as previously described (2), up to the step for adaptor ligation and purification. The purified ligation products were then equally divided into 2 …