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Open Access Technical Note

Non PCR-amplified Transcripts and AFLP®® fragments as reduced representations of the quail genome for 454 Titanium sequencing

Sophie Leroux1, Katia Feve1, Florence Vignoles1, Olivier Bouchez2, Christophe Klopp3, Céline Noirot4, David Gourichon5, Sabine Richard6, Christine Leterrier7, Catherine Beaumont8, Francis Minvielle9, Alain Vignal1 and Frédérique Pitel1*

Author Affiliations

1 UMR INRA/ENVT Laboratoire de Génétique Cellulaire, INRA, 31326 Castanet-Tolosan, France

2 Plateforme génomique (PlaGe), Génopole Toulouse-Midi-Pyrénées, INRA, 31326 Castanet-Tolosan, France

3 Sigenae UR875 Biométrie et Intelligence Artificielle, INRA, 31326 Castanet-Tolosan, France

4 Plateforme bioinformatique Genotoul UR875 Biométrie et Intelligence Artificielle, INRA, 31326 Castanet-Tolosan, France

5 PEAT, Pôle d'Expérimentation Animale de Tours, INRA, 37380 Nouzilly, France

6 Institut de Génomique Fonctionnelle de Lyon, ENS Lyon, 69364 Lyon, France

7 UMR6175 Physiologie de la Reproduction et des Comportements, INRA, 37380 Nouzilly, France

8 UR83 Recherche Avicoles, INRA, 37380 Nouzilly, France

9 UMR 1313 INRA/AgroParisTech, Génétique animale et biologie intégrative GABI, 78352 Jouy-en-Josas, France

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BMC Research Notes 2010, 3:214  doi:10.1186/1756-0500-3-214

Published: 28 July 2010

Abstract

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.

Findings

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.