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

BMC Research Notes, Jul 2010

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.

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

BMC Research Notes Non PCR-amplified Transcripts and AFLP® fragments as reduced representations of the quail genome for 454 Titanium sequencing Sophie Leroux 0 3 Katia Feve 0 3 Florence Vignoles 0 3 Olivier Bouchez 2 Christophe Klopp 1 Céline Noirot 7 David Gourichon 6 Sabine Richard 5 Christine Leterrier 4 Catherine Beaumont 9 Francis Minvielle 8 Alain Vignal 0 3 Frédérique Pitel 0 3 0 UMR INRA/ENVT Laboratoire de Génétique Cellulaire, INRA , 31326 Castanet- Tolosan , France 1 Sigenae UR875 Biométrie et Intelligence Artificielle, INRA , 31326 Castanet-Tolosan , France 2 Plateforme génomique (PlaGe) , Génopole Toulouse-Midi- Pyrénées, INRA, 31326 Castanet-Tolosan , France 3 UMR INRA/ENVT Laboratoire de Génétique Cellulaire, INRA , 31326 Castanet- Tolosan , France 4 UMR6175 Physiologie de la Reproduction et des Comportements, INRA , 37380 Nouzilly , France 5 Institut de Génomique Fonctionnelle de Lyon, ENS Lyon , 69364 Lyon , France 6 PEAT, Pôle d'Expérimentation Animale de Tours, INRA , 37380 Nouzilly , France 7 Plateforme bioinformatique Genotoul UR875 Biométrie et Intelligence Artificielle, INRA , 31326 Castanet-Tolosan , France 8 UMR 1313 INRA/ AgroParisTech, Génétique animale et biologie intégrative GABI , 78352 Jouy- en-Josas , France 9 UR83 Recherche Avicoles, INRA , 37380 Nouzilly , France 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. - Findings Next-generation sequencing can now generate from hundreds to thousands of megabases worth of data at a time [1]. Although this is a great progress when compared to conventional Sanger sequencing, it remains costly to obtain SNP by whole-genome sequencing of many individuals, especially in species for which no assembly is available. Therefore, sequencing reduced representations libraries is still an efficient and sparing approach. We describe here two protocols used for high-throughput sequencing in quail with several improvements to existing methods [2,3]. These can be applied to 454 library preparations for several purposes, such as cDNA sequencing without sample amplification, genomic DNA sequencing of scarce samples, or genomic sample multiplexing. Transcripts library preparation Total RNA was extracted from about 500 mg of adult quail brains (3 quail lines [4,5], 4 samples each) and total embryos (E8 stage, 3 quail lines, 2 samples each) according to the technique described by Le Meur et al [6], slightly modified. Briefly, tissues were homogenized with a tissue homogenizer (TH, OMNI International) with extraction solution (LiCl 3 M, urea 6 M, sodium acetate 10 mM, pH 5.6), and conserved overnight at 4°C. After centrifugation, pellets were washed twice with washing solution (LiCl 4 M, urea 8 M) and dissolved in 500 μl of TES (Tris 50 mM, EDTA 20 mM, SDS 0.5%, pH 7.5) with Proteinase K (100 μg/ml). After incubation (30 min, 37°C), total RNA was phenol extracted with Phase Lock Gel (Phase Lock Gel Heavy, 2 ml, 5 Prime) according to the manufacturer’s instructions except for the use of LiCl 5 M instead of sodium acetate 2 M. Total RNA yield was estimated on a NanoDrop 1000 spectrophotometer (NanoDrop). Next, a DNase treatment was performed to remove any potential remaining DNA. For 45 μg of total RNA per sample, 20 U of RNasine (Promega), 10 U of DNAse (Roche), 1× of PCR buffer (Invitrogen), and 1.5 mM of MgCl2 (Invitrogen) were added after which the samples were incubated 30 min at 37°C, before a purification step (RNeasy minElute Cleanup, Qiagen). A control PCR was systematically performed to check for the absence of genomic DNA. The quality of RNA samples was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Equivalent quantities of each sample were pooled to obtain 75 μg of treated total RNA, for each quail line. Poly(A)+ RNAs were then purified twice using the Dynabeads Olig (...truncated)


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Sophie Leroux, Katia Feve, Florence Vignoles, Olivier Bouchez, Christophe Klopp, Céline Noirot, David Gourichon, Sabine Richard, Christine Leterrier, Catherine Beaumont, Francis Minvielle, Alain Vignal, Frédérique Pitel. Non PCR-amplified Transcripts and AFLP®® fragments as reduced representations of the quail genome for 454 Titanium sequencing, BMC Research Notes, 2010, pp. 214, Volume 3, Issue 1, DOI: 10.1186/1756-0500-3-214