Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

Abstract

BACKGROUND Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. METHODS For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. RESULTS Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. CONCLUSIONS This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Trial by Dutch laboratories for evaluation of non‐invasive prenatal testing. Part I—clinical impact

“…Some of these laboratories already had several years of experience in fetal cfDNA analysis while others started this service especially for the TRIDENT study 27, 28, 29. Although the numbers are still limited, we found no evidence for an inferior performance of NIPT by our laboratories compared to other providers 9…”

Successful Noninvasive Trisomy 18 Detection Using Single Molecule Sequencing

“…For noninvasive detection of trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome), however, it seems to be more difficult to achieve similar results (7)(8)(9)(10). Although theoretically molecules from different regions of a genome should be sequenced uniformly by MPS, preferential amplification of sequences, depending on different guanine-cytosine (GC) content, has been observed (1,11,12 ). In contrast to the average GC content of chromosome 21, relatively low GC content occurs in chromosomes 13 and 18 (1,12 ).…”

“…Although theoretically molecules from different regions of a genome should be sequenced uniformly by MPS, preferential amplification of sequences, depending on different guanine-cytosine (GC) content, has been observed (1,11,12 ). In contrast to the average GC content of chromosome 21, relatively low GC content occurs in chromosomes 13 and 18 (1,12 ). Therefore, nonuniform amplification of these chromosomes could occur on PCR-based MPS platforms.…”

“…We previously demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias (12 ). This specific method of sequencing requires no PCR amplification step during sample preparation or during flow cell processing and results in a more pronounced noninvasive detection of trisomy 21.…”

Refining Noninvasive Prenatal Diagnosis with Single-Molecule Next-Generation Sequencing

“…In this issue of Clinical Chemistry, van den Oever et al describe the application of singlemolecule NGS (with the Helicos BioSciences platform) to the detection of trisomy 21 (11 ). NGS has developed substantially over the past 5 years, but most methods require the preliminary amplification of genomic DNA sequences, a process that is prone to sequence-specific artifacts, notably for regions of high GC content.…”