title: Optimization of multiplexed RADseq libraries using low-cost adaptors.
abstract: Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.
authors: Laboratoire de Biométrie et Biologie Evolutive, CNRS, UMR 5558, Université Lyon 1, Université de Lyon, 43 boulevard du 11 novembre 1918, 69622, Villeurbanne, France, helene.henri@univ-lyon1.fr.
authors: Hélène
authors: H
authors: Henri
authors: Marie
authors: M
authors: Cariou
authors: Gabriel
authors: G
authors: Terraz
authors: Sonia
authors: S
authors: Martinez
authors: Adil
authors: A
authors: El Filali
authors: Marine
authors: M
authors: Veyssiere
authors: Laurent
authors: L
authors: Duret
authors: Sylvain
authors: S
authors: Charlat
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics; D015723:Gene Library; D023281:Genomics / Q000191:economics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics* / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000191:economics* / Q000379:methods*