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.


CONCLUSIONS
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.
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche, Via F, Cervi 93, I-20090 Segrate, Italy. bordoni@itb.cnr.it
Roberta
R
Bordoni
Raoul
R
Bonnal
Ermanno
E
Rizzi
Paola
P
Carrera
Sara
S
Benedetti
Laura
L
Cremonesi
Stefania
S
Stenirri
Alessio
A
Colombo
Cristina
C
Montrasio
Sara
S
Bonalumi
Alberto
A
Albertini
Luigi Rossi
LR
Bernardi
Maurizio
M
Ferrari
Gianluca
G
De Bellis
D030342:Genetic Diseases, Inborn / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*