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The Effects of Restriction‐Enzyme Choice on Properties of Genotyping‐by‐Sequencing Libraries: A Study in Cassava ( Manihot esculenta )
Author(s) -
Hamblin Martha T.,
Rabbi Ismail Y.
Publication year - 2014
Publication title -
crop science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.76
H-Index - 147
eISSN - 1435-0653
pISSN - 0011-183X
DOI - 10.2135/cropsci2014.02.0160
Subject(s) - biology , restriction enzyme , genotyping , single nucleotide polymorphism , genetics , locus (genetics) , genome , manihot esculenta , population , computational biology , restriction fragment length polymorphism , genotype , dna , botany , gene , demography , sociology
Compared with other reduced‐representation sequencing methods, library construction for genotyping‐by‐sequencing (GBS) is simpler and less expensive. However, elimination of size‐selection steps results in libraries of more variable fragment size than with other reduced‐representation methods, affecting several aspects of the data. To test the effect of restriction enzyme choice on library quality, we made GBS libraries with Pst I (6‐cutter), Pst I/ Taq I (4‐cutter), or Ape KI (4.5‐cutter) from the same set of DNAs from a cassava ( Manihot esculenta Crantz) biparental population. Tag and single nucleotide polymorphism (SNP) counts were limited by the number of cut sites rather than by read number. Depth per locus was very skewed for the Pst I library, such that most SNPs had low read depth but a subset had very high read depth. In contrast, the Ape KI and Pst I/ Taq I libraries had less variable distributions of read depth and yielded far more scorable SNPs. Our results suggest that 6‐cutter enzymes may be most appropriate for genotyping a modest number of markers at a high multiplexing level, or for very large genomes, and perform better when used in a double digest with a 4‐cutter enzyme.