Rice Genetics IV - IRRI books - International Rice Research Institute

a different sequence or contig. Figure 3 shows the graphical output generatedby the Miropeats program. You can search with repeat tags or use PHRAPVIEW(part of the CONSED package [Gordon et al 1998]) to get a closer sequenceview of any possible joins suggested by Miropeats, including those that mayhave been blocked in the initial assembly by poor-quality sequence on the endsof contigs.5. Use PHRAPVIEW to check forward-reverse pairs within the assembly. Anyread pairs bridging a gap will help to order the contigs, and those within thecontigs monitor the integrity of the assembly (i.e., repeats).6. Using the primer picking function in CONSED, pick two custom primers to“walk” on subclones (or to use for polymerase chain reaction, PCR, if no appropriateclones are available) at the ends of each of the contigs. Custom primersthat are to be used for PCR or sequencing must be unique to avoid randompriming events. Primer walking will extend the sequence on the ends of theremaining contigs and facilitate joins. Remember to check for unique primersby searching the primer sequence against the rest of the assembly, to chooseprimers in the proper direction, and to choose the priming site approximately100 bases from the end of the contig, allowing a buffer zone for a poor-qualitybeginning sequence.7. Begin the first-pass edit. Generally, the first pass is the most time-consumingbecause of discrepancies that arise from the presence of one or a small numberof poor-quality sequences in an otherwise high-quality area. These areas some-0 10.0 20.0 30.0 40.0Fig. 3. Graphical output of the Miropeats program (Parsons, unpublished).Miropeats displays regions of highly similar sequence both within andamong contigs by connecting the regions with lines indicating the lengthand orientation of the sequence match.202 de la Bastide et al