On 2011-04-15, at 8:59 AM, Andrei Tudor wrote: > Hello, > > I have just finished a backbone assembly. It is the first time I have done > such an assembly, and i am wondering what do I do with the resulting files. > I saw that instead of a multifasta file with contigs, MIRA made 1 hole > chromosome from the reads. Does this mean that I do not have to creade a > pseudomolecule? > If not what should I do next? > What I usually do next is (using gap5) 1. Use tg_index to convert the CAF file to a gap5 database 2. "Top and tail" the assembly, i.e. make sure that you have true circularity if your genome is a circular one. Then using gap5, trim the start and end of the genome to make sure the coordinates match up as a circular genome. 3. Next I use the assembly view in gap5 (or use Tablet), to look for: a. holes - where there is no coverage of the corresponding region in the scaffold b. Areas of very low coverage - indicating possible misassembly, using the data and Mira's tags to look for flanking regions which can be closed by fresh PCR c. Regions of extremely high coverage - indicating repeats. I usually PCR the regions from HIGH coverage (usually in a repeat) to normal or low coverage (indicating the flanking non-repeated sequence), to make sure that scaffold-bias has not influenced the assembly. d. I pay special attention to what I call "cliffs" - regions of very high coverage next to regions of very low coverage. 4. I browse through Mira's tags using gap5 to look for areas that Mira wants me to check. The manual has good coverage of how to do that. 5. Then I scan the sequence to proofread it using gap5. I don't care about pads but I use gap5's "find next" search parameter (using the consensus quality selection) to scan and fix obvious miscalls - where there is obviously NO pad but mira has put a base there - there are not many of those. Then I am done. The ORF-calling software should find bases that could be indels causing frameshifts - let that software remove that worry! HTH John