R-042. Using Solexa Sequencing to Identify Adaptive Mutations in Methylobacterium

N. Delaney, M-C. Lee, D. Chou, C. Marx;
Harvard Univ., Cambridge, MA.

Evolving an organism for a specific challenge and then determining what genetic changes increase the fitness of that organism can provide insight into an organism’s physiology and the evolutionary process that shaped it. Although it has traditionally been difficult to determine every genetic step on an evolving organism’s adaptive walk, recent high-throughput sequencing innovations, in particular sequencing by synthesis technologies that give many small length reads may change this. In order to investigate the ability of short sequence reads to resolve genetic changes that underlie adaptation seen in experimental evolution, we undertook a theoretical and empirical evaluation of these methods using the organism Methylobacterim extorquens AM1. Computational analysis of this organism’s ~6.9 Mb genome revealed that over 96% of reads at least 20 bp long that could possibly be generated by the reference organism map to a unique genomic location. Additionally, we show that at sufficient coverage levels short read sequencing are highly likely to capture all the mutations that separate an evolved strain from its ancestor. To test these predictions, we evolved populations of M. extorquens for analysis by short read sequencing. 5 populations were evolved for ~600 generations to adapt to diverse physiological challenges. Following this, an isolate from each population was sequenced using Solexa machines that generated 36 bp sequence reads at over 15X coverage. We show that the Solexa sequencing data was able to discover many physiologically important mutations that were independently verified by PCR amplification, and that the data produced are in good agreement with computational predictions. We conclude that short read sequencing technology is a useful method to allow evolutionary changes to inform physiology studies.