Rice Genetics IV - IRRI books - International Rice Research Institute

sequencing method for us on cDNA clones for which oligo walking was both costlyand time-consuming because of the turnover time of ordering oligos and primer failures.The linking of universal primer sequences to both sides of the transposon allowssequencing to be done in a “shotgun” fashion on multiple clones, thereby vastly expeditingthe coverage of the problem region (see Fig. 6).RepeatsRepeats may cause physical and/or sequence gaps and are often difficult to detect.In the human sequence, ALUs are small, frequent repeats that are, on average, 200–300bases in length. However, repetitive elements may be quite large in size and maydiffer by as little as one base in several kilobases of sequence. Careful inspection andinterpretation of the sequence data during editing and assembly are imperative lestrepeats be misassembled. Restriction digests are often clues to improper assembly andrepeat problems. The various types of repeats and some strategies to resolve them follow:1. Direct repeats—may have a unique sequence between the repetitive elements.Try primer walking, long reads, etc., to walk into the unique area. Physical+Template containingan area of secondarystructureTransposonsRandom transposoninsertionSecondary structuredisrupted by transposoninsertionFig. 6. Schematic of the in vitro transposon sequencing strategy. Insertion of atransposon flanked by two universal priming sites into clones containing regionsof secondary structure can often help resolve difficult sequence regions by disruptingthe secondary bonding.208 de la Bastide et al