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

Identifiers: SRA: SRP010776
Study Type: 
Population Genomics
Abstract: 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.
Description: An inexpensive and practical method for measuring variation in single individuals has multiple applications including measuring the effect of mutagenic treatments. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference.

Related SRA data

8 ( 4 samples )
8 (8.6Gbp; 5.3Gb)