SEQUENCE ASSEMBLY

The figure illustrates the use of GDE for combining sequencing reactions into a single contig.

1. The raw sequence from each sequencing reaction was read in from the File menu (ebf85 etc.).
2. Flanking vector sequences were removed manually
3. Only one strand of each reaction is needed. Where reactions are opposite to the reading frame,
   1. sequence is selected
   2. DNA/RNA -> Complement to generate complementary strand
   3. Edit -> Reverse to flip sequence (restores 5'->3' orientation)
   4. File -> Get Info to add "-opp" to the name. This is a good way of documenting the fact that the original reaction was on the other strand.
4. To assist in assembling the sequence, comments are added in the top two lines showing the locations of PCR primers and exon/intron boundaries. Gaps are inserted by hand to align raw sequences.
5. The composite sequence is generated by selecting all raw sequences and choosing Alignment -> Sequence consensus.
6. For verification purposes, the consensus is translated in 3 reading frames, preserving codon spacing (DNA/RNA -> Translate). In the figure, reading frames 2 & 3 are the reading frames of the 1st and 2nd exons. Reading frame 1 was deleted from the alignment.
7. The consensus is saved to a separate file using the File menu.

The final sequence file could be read using SEQUIN to prepare a submission to GenBank.