Table 1.
Amplification/Sequencing Primers and Amplicon Electrophoretic Mobility Evaluation.
Table 2.
ND132 Template Amplification Summary.
Table 3.
Sequences of the Six D. desulfuricans ND132 Gaps.
Figure 1.
Example of the increase in secondary structure following determination of the gap sequences: Gap 1.
Left- secondary structure prediction done using the Mfold web server [29] to aid in primer site selection. Closed triangle shows the location of the gap. Structure was prepared by appending 100 nt at the termini of the 5′ and 3′ contigs surrounding the gap. Folding parameters used 50 mM NaCl, and 2.5 mM MgCl2 at 60°C. Excess nucleotides were trimmed from the structure for presentation. Right – secondary structure including the determined gap sequence. The determined gap sequence nucleotides are highlighted in upper case. Folding parameters are as given for the structure prepared prior to gap sequence determination.
Table 4.
Survey of Additional Non-contiguous Finished Genomes.
Figure 2.
Top- 100 nt from either side of the gap are appended and used for alignment search to identify and potential problems with assembly causing the gap. N(n) denotes unknown nucleotides (50 or 100 by convention) inserted into the gaps in deposited sequence data The resulting 200 nt artificial contig is subjected to 2° structure analysis. The folding analysis reveals additional 2° structures in the vicinity of the gap that may present added difficulty with amplification and sequencing. Primers are targeted to positions proximal to the gap relative to any additional problems identified in the folding evaluation. Where the gap position is involved with secondary structure, amplification with an SD polymerase and use of a two-step extension cycle (1 min at 72°C followed by 1 min at 75°C) supports amplification. The second segment of the extension cycle can be modified based on the thermal stability identified in the initial folding analysis.