I am a student in Plant phylogenetics. I am using the Staden Package to assemble my tracerfiles into a consensus sequence. The tracerfiles come from a direct cycle sequence, so I have a forward and a reverse tracerfile.
Shotgun assembly in Pregap4 1.5 is working fine for genes that I have amplified in one piece. For large genes (e.g. the chloroplast gene rbcL), amplification was in two pieces. Hence I have four tracerfiles. There is some overlap in the two internal primers (aprox. 60 bp.), but Shotgun will not assemble them apropriately.
I tried fiddling with the Shotgun parameters match, pads and mismatch, but without any result.
If anyone has experiences with such an assembly, or knows it to be impossible to assemble with such a small overlap, please let me know.
thanks in advance, Robin v. Velzen
Try using find internal joins to look into the cutoff data too. The overlap may to too poor quality to detect using "good" data alone.
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