Open Access Highly Accessed Methodology article

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

Roberta Bordoni1, Raoul Bonnal1, Ermanno Rizzi1, Paola Carrera23, Sara Benedetti23, Laura Cremonesi23, Stefania Stenirri23, Alessio Colombo3, Cristina Montrasio3, Sara Bonalumi23, Alberto Albertini1, Luigi Rossi Bernardi1, Maurizio Ferrari234 and Gianluca De Bellis1*

Author Affiliations

1 Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche (CNR-ITB), Via F. Cervi 93, I-20090 Segrate, Italy

2 Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milan, Italy

3 Laboratory of Clinical Molecular Biology, Diagnostica e Ricerca San Raffaele S.p.A., Milan, Italy

4 Vita-Salute San Raffaele University, Milan, Italy

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BMC Genomics 2008, 9:464  doi:10.1186/1471-2164-9-464

Published: 8 October 2008



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.


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 30× depth were correctly recognized.


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.