Andolfatto_1
Multiplexed Shotgun Genotyping for Rapid and Efficient Genetic Mapping
101 base pair reads from a reduced representation library. we prepared a multiplexed shotgun library of 96 progeny (48 males and 48 females) from an F1 backcross experiment. Specifically, D. simulans w501 was crossed to D. sechellia w1. These closely-related species can be crossed to generate fertile F1 females and sterile F1 males (Lachaise et al. 1986). The F1 females were subsequently backcrossed to males of the D. sechellia w1 parental strain. The genomes of the two parental species are approximately two percent divergent at the nucleotide level(Kliman et al. 2000). 10 ng of genomic DNA from each individual was digested with the restriction enzyme MseI and 96 custom-designed adapters, each carrying a unique 6 bp barcode, were ligated to the restriction fragments (Figure 1). The ligation reactions were combined, fragments were size-selected, and standard Illumina flow-cell adaptors were added using PCR with custom designed primers. Sequencing this library produced approximately 22 million 101 bp reads in a single lane of an Illumina Genome Analyser. Sequences were parsed into 96 groups—representing the 96 backcross progeny based on a 6 base-pair barcode present at the beginning of each read.
We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kilobases of its true position using 96 F1- backcross individuals genotyped in a single lane on an Illumina Genome Analyser. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38 kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.