All those conforming, at least in part, to the selected templates (1kil, 1s35, 1sum, 1txd, 2boq, 2i0m, 2of3) were used in further analysis, along with five models produced by PROTINFO [40] from the two full length templates (1sum, 2i0m).
The resulting collection of 14,227 sequences represented my full-length template database.
On average the resulting alignments were between 0.61% worse and 0.34% better than the alignments generated by SILVA when using full-length template sequences.
Thus, the replicated products were predominantly the same length as the full-length template.
Only 22 transmembrane beta-strands were detected when the full-length template sequence was analyzed.
To determine the specificity of the reactions, all reaction conditions were tested against a purified full-length template as well as other non-specific splice variants.
When searching against full-length templates, 7 to 9-mers provided the closest matches.
To assemble the full-length templates, the PCR products encoding Leishmania Rabs were fused by PCR with the universal fragment 5 (Fig. S2A), while the fragments containing mammalian Rab were fused with fragment 6 (Fig. S2D).
Thus, there are no pre-existing full-length templates to align the sequencing reads.
The parental background could arise either from contamination of the PCR reaction with full-length templates, or by the accidental elongation of the unpaired extension on a strand containing no crossovers (skew extension without recombination [ 41] via template switching).
The composition of PCR mixture included (per 50 µl) Qiagen PCR buffer (1.5 mM MgCl2, KCl, (NH4 2SO4, tris-HCl, pH 8.7), 1.5 mM MgCl2, 200 µM dATP, dCTP dGTP, 70 µM dTTP, 20 µM dUTP-Cy3, 80 nM or forward primer, 1 µM of reverse primer, 1.25 U of Qiagen Hzot-Start Taq-polymerase, and 1 µl of full length amplicon template.
