SEQUENCE ALIGNMENT USING BIOEDIT
BioEdit is free for use by any and all interested parties, but is no longer being regularly maintained. The
program can be downloaded from http://www.mbio.ncsu.edu/bioedit/bioedit.html
a. Open the file sequences.fasta in BioEdit
b. If necessary, you can then File>Import>Sequence alignment file to add sequences_single.fasta to the alignment.
c. Make sure Mode: is set to Edit using the dropdown menu.
d. Another dropdown menu will become visible to the right of the Edit dropdown. Make sure this is set to Insert.
e. Sequences that have ended up in the FASTA file in the wrong orientation (insect sequences should typically start AAC or TAC) may be corrected by highlighting the sequence name by clicking the cursor on it, clicking the Sequences menu at the top of the screen. Moving the cursor down the dropdown to Nucleic Acid and clicking Reverse complement.
f. Sequences all need to be 658 bp and aligned to each other (before proceeding for further analysis or before uploading to BOLD and GenBank). This can be done by typing additional Ns at the beginning and end of the sequences in the BioEdit Edit mode. Be sure to check across the whole of the alignment of the sequences that you have added the correct number of Ns.
g. In the figure below featuring a 50-bp barcode for simplification, LOP001-11 is of full length. LOP002-11 needs 6 Ns adding to the left side of the sequence to become aligned, while LOP003-11 needs 4 Ns adding to the right side of the sequence to be 50 bps long. LOP005-11_F needs to be reverse complemented to be in the same orientation as the other sequences.
h. Sequences which were not part of a consensus (i.e., when one direction failed but the single sequence is of sufficient length and quality for submission to BOLD) may appear in the FASTA still tagged with the direction. This needs to be deleted, e.g., the sequence named LOP005-11_F should be renamed as LOP005-11.
i. If you are having trouble with the alignment, a good quality (i.e., 658[0n]) sequence can be downloaded from BOLD and imported into your BioEdit file as a guide, e.g., MHAHC824-05 (Fig. 3 ). Be sure to delete this sequence before saving the file.
j. Save the file (File > Save).
program can be downloaded from http://www.mbio.ncsu.edu/bioedit/bioedit.html
a. Open the file sequences.fasta in BioEdit
b. If necessary, you can then File>Import>Sequence alignment file to add sequences_single.fasta to the alignment.
c. Make sure Mode: is set to Edit using the dropdown menu.
d. Another dropdown menu will become visible to the right of the Edit dropdown. Make sure this is set to Insert.
e. Sequences that have ended up in the FASTA file in the wrong orientation (insect sequences should typically start AAC or TAC) may be corrected by highlighting the sequence name by clicking the cursor on it, clicking the Sequences menu at the top of the screen. Moving the cursor down the dropdown to Nucleic Acid and clicking Reverse complement.
f. Sequences all need to be 658 bp and aligned to each other (before proceeding for further analysis or before uploading to BOLD and GenBank). This can be done by typing additional Ns at the beginning and end of the sequences in the BioEdit Edit mode. Be sure to check across the whole of the alignment of the sequences that you have added the correct number of Ns.
g. In the figure below featuring a 50-bp barcode for simplification, LOP001-11 is of full length. LOP002-11 needs 6 Ns adding to the left side of the sequence to become aligned, while LOP003-11 needs 4 Ns adding to the right side of the sequence to be 50 bps long. LOP005-11_F needs to be reverse complemented to be in the same orientation as the other sequences.
h. Sequences which were not part of a consensus (i.e., when one direction failed but the single sequence is of sufficient length and quality for submission to BOLD) may appear in the FASTA still tagged with the direction. This needs to be deleted, e.g., the sequence named LOP005-11_F should be renamed as LOP005-11.
i. If you are having trouble with the alignment, a good quality (i.e., 658[0n]) sequence can be downloaded from BOLD and imported into your BioEdit file as a guide, e.g., MHAHC824-05 (Fig. 3 ). Be sure to delete this sequence before saving the file.
j. Save the file (File > Save).