Genetica

, Volume 143, Issue 2, pp 139–143 | Cite as

Optimization of multiplexed RADseq libraries using low-cost adaptors

  • Hélène Henri
  • Marie Cariou
  • Gabriel Terraz
  • Sonia Martinez
  • Adil El Filali
  • Marine Veyssiere
  • Laurent Duret
  • Sylvain Charlat
Article

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.

Keywords

Reduced representation genomics RADseq Protocol Multiplexing 

Notes

Acknowledgments

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.

Supplementary material

10709_2015_9828_MOESM1_ESM.xlsx (37 kb)
S1: This table provides a list of the 755 MIDs generated by the barcrawl program. (XLSX 37 kb)
10709_2015_9828_MOESM2_ESM.xlsx (14 kb)
S2: This table provides a list of the 100 MIDs used in our study. (XLSX 14 kb)
10709_2015_9828_MOESM3_ESM.doc (42 kb)
S3: Step by step protocol for the preparation and utilization of P1 adaptors for highly multiplexed RAD sequencing. (DOC 41 kb)
10709_2015_9828_MOESM4_ESM.xls (54 kb)
S4: This table provides all details concerning the DNA templates used in this study and corresponding RAD sequencing results. (XLS 54 kb)
10709_2015_9828_MOESM5_ESM.xlsx (10 kb)
S5: This tables provides the number of loci and reads from control samples mapping to the D.melanogaster genome. (XLSX 9 kb)

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Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Hélène Henri
    • 1
  • Marie Cariou
    • 1
  • Gabriel Terraz
    • 1
  • Sonia Martinez
    • 1
  • Adil El Filali
    • 1
  • Marine Veyssiere
    • 1
  • Laurent Duret
    • 1
  • Sylvain Charlat
    • 1
  1. 1.Laboratoire de Biométrie et Biologie Evolutive, CNRS, UMR 5558, Université Lyon 1Université de LyonVilleurbanneFrance

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