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.
Bordoni
Roberta
R
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche, Via F, Cervi 93, I-20090 Segrate, Italy. bordoni@itb.cnr.it
Bonnal
Raoul
R
Rizzi
Ermanno
E
Carrera
Paola
P
Benedetti
Sara
S
Cremonesi
Laura
L
Stenirri
Stefania
S
Colombo
Alessio
A
Montrasio
Cristina
C
Bonalumi
Sara
S
Albertini
Alberto
A
Bernardi
Luigi Rossi
LR
Ferrari
Maurizio
M
De Bellis
Gianluca
G
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*