Rice Genetics IV - IRRI books - International Rice Research Institute

times contain sufficient data to make editing changes without requiring additionalreactions. Always check the primary chromatogram data as well as thePHRED quality values of the available sequence before making an edit. Thefirst pass is also the first attempt to resolve sequence ambiguities that do requireadditional data (i.e., compressions, low-quality areas, and single-cloneareas). In problem areas that require more sequencing data, it is important toemploy an aggressive finishing strategy to maximize efficiency. An aggressiveapproach is one in which multiple templates (2–5) are called to resolve a givenproblem so that, if one reaction fails, another will be available to take its place.Additionally, in an aggressive approach, custom primers would be ordered tocover single-clone areas, rather than trying to extend reads that may not belong enough to cover the area. Multiple custom oligos should be ordered toextend contigs or cover problem areas in case the first oligo does not work.Remember to tag any problem areas (i.e., compressions, repeats, clone variations,single base runs in which the number of bases varies, etc.) so that youcan easily return to the area for study. Do not remove tags even if the problemis resolved! During the first-pass edit, it is a good idea to intermittently performsome of the sequencing reactions generated by editing to allow extensionreactions to be entered into the database early in the finishing process. Earlyaccess to this data may expedite joins, reduce turnaround time of orderingadditional primers for PCR or subclone walking if the first set fails, or elucidateBAC insert/vector junctions or read pairs that will help to order contigs.8. After the first-pass editing is complete and all of the first-pass reactions havebeen assembled into the project, a second editing round begins. The secondediting pass is more aggressive than the first round in several ways. Templatesmay be regrown to fix problem areas, custom oligos that may be used for PCRor subclone walking are ordered for any regions still in need of coverage, andPCR is done for any remaining gaps including vector junctions. This finishinground should resolve the majority of discrepancies and leave only a small numberof problem areas for the third (and, we hope, final) edit. In the third-passedit, more complicated techniques such as transposon insertion or small insertlibraries may be employed to resolve any remaining gaps, repeats, or othersecondary structures (see the next section, “Techniques for problem areas”).9. The final check consists of checking the entire clone for contiguity, consensusquality (PHRED scores), coverage, vector junctions, and the proper resolutionof repeats. Tagged regions should be checked to ensure that the problem hasbeen sufficiently corrected. Single-stranded, single chemistry regions must havea minimum quality of PHRED30 at each base. The clone should be recheckedfor single-clone regions and any areas lacking coverage. Gaps must be filledwith a sufficient amount of good-quality data and coverage. Regions coveredonly by PCR must be amplified with a high-fidelity enzyme and must be taggedfor annotation. Transposon sequences must be excised and theregion must be tagged for annotation. Vector junctions must contain a restrictionsite and must be tagged as a BAC cloning vector. Any other region need-Strategies and techniques for finishing genomic sequence 203