Optimization of multiplexed RADseq libraries using low-cost adaptors
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.
KeywordsReduced representation genomics RADseq Protocol Multiplexing
This work was funded by the Centre National de la Recherche Scientifique (ATIP Grant to SC) and the Agence Nationale de la Recherche (Grant ClimEvol). GT is the recipient of a Ph.D. studentship from the Rhône-Alpes region (“Program Cible” Grant). We would like to thank the two anonymous reviewers for their critical assessment of our work.
Conflict of interest
The authors declare that they have no conflict of interest.
- Etter PD, Bassham S, Hohenlohe PA et al (2011) SNP discovery and genotyping for evolutionary genetics using RAD sequencing. In: Orgogozo V, Rockman MV (eds) Molecular methods for evolutionary genetics. Humana Press, Totowa, pp 157–178Google Scholar
- Sambrook J, Russel D (2001) Commonly used techniques in molecular cloning. In: Sambrook J, Russel D (eds) Appendix 8, in molecular cloning, vol 3, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar