Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer.

BACKGROUND: A new priority in genome research is large-scale resequencing of genes to understand the molecular basis of hereditary disease and cancer. We assessed the ability of massively parallel pyrosequencing to identify sequence variants in pools. From a large collection of human PCR samples we selected 343 PCR products belonging to 16 disease genes and including a large spectrum of sequence variations previously identified by Sanger sequencing. The sequence variants included SNPs and small deletions and insertions (up to 44 bp), in homozygous or heterozygous state. RESULTS: The DNA was combined in 4 pools containing from 27 to 164 amplicons and from 8,9 to 50,8 Kb to sequence for a total of 110 Kb. Pyrosequencing generated over 80 million base pairs of data. Blind searching for sequence variations with a specifically designed bioinformatics procedure identified 465 putative sequence variants, including 412 true variants, 53 false positives (in or adjacent to homopolymeric tracts), no false negatives. All known variants in positions covered with at least 30x depth were correctly recognized. CONCLUSION: Massively parallel pyrosequencing may be used to simplify and speed the search for DNA variations in PCR products. Our results encourage further studies to evaluate molecular diagnostics applications.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.

The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

Using comparison scenarios to improve prenatal risk communication.

UNLABELLED: The present research provides empirical evidence of whether communicating the prenatal risk of chromosomal anomalies using comparison scenarios influences women's ability to distinguish between different risk levels. In 2 experiments, participants read a description of a hypothetical woman who was learning of the risk of chromosomal anomaly as a result of a prenatal screening test. Both experiments used a 3 (risk level) × 3 (scenario) full between-subjects design. In accordance with the experimental condition, participants were presented with a low (e.g., 1 in 5390), a medium (e.g., 1 in 770), or a high risk value (e.g., 1 in 110). Such risk values were presented either on their own or along with additional information illustrating a comparison scenario that provided 2 numerical comparison points. Participants were asked to evaluate the risk of chromosomal anomaly. In Experiment 2, participants' numeracy skills were also assessed. RESULTS: showed that the use of comparison scenarios results in significant differences in perceived risk across risk levels whereas such differences are not significant without the comparison scenario, but such a technique has differential effects according to participants' capacity to deal with numbers. Although the technique is beneficial for high-numerate participants, it has no effect on low-numerate participants.