Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

We describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modi- fied to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method using rice and arabidopsis. To test its suit- ability for the discovery of very rare SNP, one control and three mutagenized rice indi- viduals (1, 5 and 10 mM sodium azide) were used to prepare genomic libraries for Illumi- na sequencers by ligating barcoded adapters to NlaIII restriction sites. For genome- dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15X. We identi- fied high-confidence base changes by comparing sequences across individuals and identi- fied instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70- mers were extracted from the sequence of the control individual and single-copy se- quence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This de novo "genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected muta- tion densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment.