Non PCR-amplified Transcripts and AFLP fragments as reduced representations of the quail genome for 454 Titanium sequencing.
BACKGROUND
SNP (Single Nucleotide Polymorphism) discovery is now routinely performed using high-throughput sequencing of reduced representation libraries. Our objective was to adapt 454 GS FLX based sequencing methodologies in order to obtain the largest possible dataset from two reduced representations libraries, produced by AFLP (Amplified Fragment Length Polymorphism) for genomic DNA, and EST (Expressed Sequence Tag) for the transcribed fraction of the genome.


RESULTS
The expressed fraction was obtained by preparing cDNA libraries without PCR amplification from quail embryo and brain. To optimize the information content for SNP analyses, libraries were prepared from individuals selected in three quail lines and each individual in the AFLP library was tagged. Sequencing runs produced 399,189 sequence reads from cDNA and 373,484 from genomic fragments, covering close to 250 Mb of sequence in total.


CONCLUSIONS
Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements.The protocols may be used for several sequencing applications, such as de novo sequencing, tagged PCR fragments or long fragment sequencing of cDNA.
Leroux
Sophie
S
UMR INRA/ENVT Laboratoire de Génétique Cellulaire, INRA, 31326 Castanet-Tolosan, France. frederique.pitel@toulouse.inra.fr.
Feve
Katia
K
Vignoles
Florence
F
Bouchez
Olivier
O
Klopp
Christophe
C
Noirot
Céline
C
Gourichon
David
D
Richard
Sabine
S
Leterrier
Christine
C
Beaumont
Catherine
C
Minvielle
Francis
F
Vignal
Alain
A
Pitel
Frédérique
F