TY - JOUR
T1 - Optimization of multiplexed RADseq libraries using low-cost adaptors
AU - Henri, Hélène
AU - Cariou, Marie
AU - Terraz, Gabriel
AU - Martinez, Sonia
AU - El Filali, Adil
AU - Veyssiere, Marine
AU - Duret, Laurent
AU - Charlat, Sylvain
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - Animals
KW - Drosophila melanogaster
KW - Gene Library
KW - Genomics
KW - High-Throughput Nucleotide Sequencing
KW - Sequence Analysis, DNA
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1007/s10709-015-9828-3
DO - 10.1007/s10709-015-9828-3
M3 - Article
C2 - 25666920
SN - 0016-6707
VL - 143
SP - 139
EP - 143
JO - Genetica
JF - Genetica
IS - 2
ER -