Abstract

Underlying chromosomal copy number variation is responsible for 10–15% of fetal structural abnormalities, but even in the presence of a normal karyotype neonatal outcome can be poor. Current whole genome prenatal analysis is limited to G-banded karyotyping. Prenatal microarrays have yet to be fully established, but detection rates of up to 5.8% are reported.1 Potential disadvantages of arrays include the reliance on nucleic acid hybridisation, where fluctuations may increase experimental noise and reduce reproducibility.2 Prenatal material is commonly of low quantity and quality, characteristics which inhibit performance of microarrays.

We have developed an alternative method of copy number diagnosis using Next Generation Sequencing (CNV-Seq). Massively-parallel sequencing of test and control individuals is performed using the Illumina Genome Analyser platform. Millions of genomic reads are aligned to the reference sequence and analysed using custom-designed segmentation algorithms. Comparison with similarly-segmented normal controls is then performed for accurate variant detection.

Analysis of platform performance showed 100% detection rate in 10 prenatal samples with known chromosomal abnormalities ranging in size from submicroscopic microdeletion syndromes to aneuploidy. Multiplexing of up to 15 samples is possible for cost benefits without loss of sensitivity. Depth of coverage required is fractions of that reported previously.3 Comparison with array CGH data supports the hypothesis that CNV-Seq offers advantages over both conventional karyotyping and DNA microarrays.

We suggest that this approach is unique in its potential to offer a single platform solution for genetic analysis, allowing detection of a range of abnormalities from single point mutations to balanced rearrangements and aneuploidy.