Suppose that we want to obtain the phylogeny of the genus Bactrocera (the genus to which the pest species Bactrocera invadens belongs to) based on all COI (cytochrome oxidase subunit I) gene sequences available at NCBI.

1. The first step is to obtain the multi FASTA file with the relevant sequences:
   1. Go to http://www.ncbi.nlm.nih.gov/ and Log in.
   2. Choose the Nucleotide database and writing in the search bar: "Bactrocera"[Organism] AND "COI"[Gene] NOT "Genome". The NOT "Genome" part of the expression will avoid downloading entire mitochondrial genomes.
   3. Download all sequences choosing the Send to File option, and selecting FASTA as format.
   4. The resulting 3.4 Mb file¹ can be imported to BDBM (using the File → Import FASTA operation or by directly placing the file into /fasta/nucleotides folder of the repository) and inspected (double click on the file under the folder FASTA Files).
   
2. Note that the definition line is long, that there are blank lines between sequences and line breaks within sequences. These issues may prevent the use of this file by the researchers' favorite software package. All these issues can be fixed using the Operations → Reformat Fasta operation.
   
   In this operation you will find the following options:
   • Sequence Fragment Length: Choosing a negative or zero sequence fragment length will remove all line breaks in the nucleotide sequences, while a positive number, for instance 60, will place a line break every 60 nucleotides (an option that may be useful when preparing FASTA files to be deposited elsewhere).
   • Renaming Mode:
     • NONE: sequence name will remain unchanged.
     • SMART: automatically extracts the gi (general identifier) codes.
     • GENERIC: extracts the information present in the fields that are delimited by the | symbol (the first field is 0).
     • PREFIX: allows the incorporation of a prefix into all sequence names with the possibility of erasing or keeping the headers.
   

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¹ On the 9^th of June 2016, 3930 COI sequences could be retrieved.

Suppose that we want to prepare a FASTA file to build a phylogeny of the SFBB F-box gene family of Fragaria vesca (strawberry). This may seem a challenging task, since there is no annotation for this species. Nevertheless, this is quite simple using BDBM.

1. Download the Fragaria vesca genome FASTA file from here (mirror).
2. Extract the FASTA file from the compressed package.
3. Import the downloaded Fragaria versca FASTA file to BDBM using the File → Import FASTA option (or copy the file to the /fasta/nucleotides folder of the repository).

4. Select the Operations → Get ORF (EMBOSS) operation and use the default parameters (we can use this option since, in plants, SFBB genes are always intronless).
   Give the name of your choice to the output (such as FraVesGETORF, the name used in this example). The output ORFs file will appear in the FASTA files folder of the repository tree.
   The new file will contain all the ORFs that start with a methionine and that end with a codon or that have no stop codons untils the end of the sequence.

5. Build a BLAST-formatted database by right clicking on top of the FraVesGETORF file and by choosing the Make Blast Database operation. Choose a name for this nucleotide database, such as FraVesGETORFDB.




6. Download the Malus (AB539857.1) protein sequence from here (mirror).
7. Remove the first 50 amino acids from the Malus (AB539857.1) protein sequence using your favourite plain text editor.

8. Import the edited protein sequence FASTA file to BDBM using the File → Import FASTA operation (or copy the file to the /fasta/proteins folder of the repository).

9. Perform a tblastn search using the BLAST → TBLASTN (with external query) operation. Use as query the edited SFBB FASTA file, and give a name to the output. Two files are generated: the BLAST output file and a file with the sequences that show a hit in the BLAST output file (the SFBB-like sequences). Both are found in the folder Exports.

For many species, such as D. melanogaster, more than one coding sequence is described for each gene due to the use of alternative translation starts, and/or alternative splicing. For instance, for the D. melanogaster Sod gene, two coding sequences are available in FlyBase (462 and 504 bp long; link). Nevertheless, in the the closely related species D. simulans, only the 462 bp long coding sequence is described in FlyBase (link). BDBM can be used to quickly obtain the other form.

1. First, we need to download the D. simulans genome assembly from here (mirror) and extract the contents.
2. Then, download the D. melanogaster 504 bp Sod isoform from here (mirror).
3. Import both FASTA files into BDBM using the File → Import FASTA option (or copy the file to the /fasta/nucleotides folder of the repository folder).
4. Running the Operations → Splign-Compart operation will produce a FASTA file with the D. simulans 504 bp isoform.

The availability of a genome annotation does not mean that CDSs are correctly annotated. When a gene annotation is very different in two closely related species, the Operations → Splign-Compart operation can be used to try to find an alternative gene annotation that would make the two gene annotations more similar.

In order to determine the feasibility of this approach, we can attempt to obtain a gene annotation for all 42 genes under the GO category germ cell development (i.e. alien, aret, bam, bgcn, Cul2, Cul5, CSN1b, CSN3, CSN4, CSN5, CSN6, CSN7, dpp, ecd, EcR, egh, fmr1, fs(1)Yb, gcl, gus, hyd, Jafrac1, Kay, lok, meiP26, nclb, Nop60B, nos, orb, osk, otu, out, pgc, pum, shg, SmB, Sxl, Tre1, tud, usp, wun2, and zpg) from 15 Drosophila genomes/transcriptomes (i.e. D. simulans, D. sechellia, D. yakuba, D. erecta, D. ficusphila, D. eugracilis, D. biarmipes, D. takahashii, D. suzukii, D. serrata, D. elegans, D. rhopaloa, D. kikkawai, D. ananassae, and D. bipectinata).

All 42 D. melanogaster protein coding genes under the GO category germ-cell development were retrieved from Flybase. For your convenience, you can download them all from here.

All but four (Jafrac1, nclb, pgc and usp) of these genes show introns in the coding region of the transcript studied. Therefore, in order to retrieve the corresponding CDS isoforms from the 15 non-melanogaster genomes and 1 transcriptome here considered, FASTA formatted genomes were downloaded from Flybase or from NCBI. You can find a list of the original URLs here, but, for your convenience, we have created a bundle with all the FASTA files, that you can download from here.

1. Download the 42 D. melanogaster protein coding genes described before.
2. For your convenience, you can download the CDS isoforms from the 15 non-melanogaster genomes and 1 transcriptome bundle instead of using the original URLs.
3. Extract the FASTA files from the compressed files.
4. Import all the FASTA files to BDBM using the File → Import FASTA operation (or copy the files to the /fasta/nucleotides folder of the repository).

5. Run the Operations → Splign-Compart using one of the 15 non-melanogaster genomes and 1 transcriptome and the multi FASTA file with a single D. melanogaster CDS isoform for each of these 42 genes.

6. Repeat the previous step for each of the 15 non-melanogaster genomes and 1 transcriptome.

Note

As noted above, when a CDS annotation is performed, the original name of the sequence is replaced by the name of the sequence that is used to obtain a given CDS. This means that it is possible to use multiple CDSs at the same time as references, but on the other hand it means that in order to keep track of the species of origin, each genome/transcriptome must be used separately and the resulting CDSs prefixed with the species name using the prefix renaming mode from the Operations → Reformat FASTA operation.

7. Create a BLAST database for each of the 16 resulting FASTA files using Operations → Make BLAST Database.

8. Run Operations → BLAST DB Alias to create and alias and treat all those databases as a single one.


9. To be able to make a TBLASTX search with each of the 42 D. melanogaster protein coding genes we need to extract each one from the FASTA file D_mel_sequences.fasta. You can achieve this by using the Operations → Retrieve Search Entry operation or your favourite text editor.

   Alternative 1. Using the Operations → Retrieve Search Entry operation

   1. Create a BLAST database for the D_mel_sequences.fasta file using Operations → Make BLAST Database.
   2. Open D_mel_sequences.fasta file doing double click in the name of the FASTA file in the repository, select the name of one of the sequences and copy it to the clipboard.
   
   3. Run Operations → Retrieve Search Entry operation over the database corresponding to the D_mel_sequences.fasta FASTA file and use sequence name copied in the previous step.
   
   4. A new Search Entry will be created in BDBM with the contents of the selected sequence.
   
   5. Repeat the previous steps for each of the 42 D. melanogaster protein coding genes.
   6. Perform a BLAST → TBLASTX search over the aliased database created in the previous steps using as query each search query created.
   
   Alternative 2. Using your favourite text editor

   1. Open the D_mel_sequences.fasta FASTA file using your favourite editor.
   2. Copy each of the 42 D. melanogaster protein coding genes and paste them into separate FASTA files.
   
   3. Perform a BLAST → TBLASTX with external query search over de DB Alias created in the previous steps usign as query each of the files created in the previous step.
   
10. Check the resulting FASTA files to see if the CDS sequence is incomplete, or if there are obvious erroneous CDS annotations due to sequencing errors.

BDBM offers an easy to use local BLAST graphical interface. While it is true that researchers must download the FASTA files of interest from existing databases and this is an extra inconvenient step relative to the existing online BLAST databases, it is also true that, in general, local BLAST searches are faster and that a much higher level of control regarding the parameters used in the searches can be achieved. Particularly, BDBM BLAST operations include an Additional parameters field that allows invoking all the arguments available for BLAST, making it similar to running blast on the computer console, but with the extra of automatically retrieving the sequences that show a hit in the blast search. For instance:

1. We could use the SFBB F-box gene annotation FASTA file produced in the Use Case 2 (Follow the instructions in Use Case 2 or download it from here).
2. Import the FASTA file to BDBM using the File → Import FASTA operation (or copy the file to the /fasta/nucleotides folder of the repository).

3. Build a BLAST-formatted database by right clicking on top of the UC2SFBB.fasta file and by choosing the option Make Blast Database.
   Choose a name for this nucleotide database, such as UC2SFBBDB.




4. Download the Malus (AB539857.1) protein sequence used in the Use Case 2 the from here (mirror).
5. This time, remove all but the first 50 amino acids (the F-box motif) from the Malus (AB539857.1) protein sequence using your favourite plain text editor.

6. Perform a tblastn search over the nucleotide database (UC2SFBBDB) using the BLAST → TBLASTN (with external query) operation. Use as query the edited SFBB FASTA file in the previous step, and give a name to the output.
   Two files are generated: the blast output file and a file with those entries that have a hit with the F-box motif, that are those that have at least a partial F-box region. Both are found in the folder Exports of the repository tree.

When performing phylogenetic analyses, researchers often concatenate sequences from different genes to increase the power to make inferences. Therefore, BDBM offers the possibility to concatenate sequences in FASTA format, as long as the sequence headers of the sequences to be concatenated are identical.

As an example of how easy and quick you can achieve this, we are going to merge 27 gene sequences of 6 different species of the Drosophila genus: D. melanogaster, D. sechellia, D. simulans, D. erecta, D. yakuba, and D. ananassae.

1. Download the gene sequences from here.
2. Extract the gene sequence files from the compressed file.
3. Copy the extracted files to the /fasta/nucleotides folder of the repository or import them using the File → Import FASTA operation.
4. Launch the Operations → Merge FASTA and select all the sequences. Remember that you can select all the sequences in the list with the Ctrl + A or Cmd + A combination and that you can select or unselect single sequences pressing Ctrl or Cmd while clicking with the mouse.

5. Once you have selected all the sequences you want to merge, launch the execution by pressing the Ok button. The resulting FASTA file will contain six sequences, one for each species.

The ProSplign/ProCompart (NCBI) option is an alternative to Splign-Compart (NCBI). When using this option, protein reference sequences rather than CDSs (nucleotide) reference sequences are used. Since protein sequences change at a slower pace than nucleotide sequences, in principle the reference and target sequences can be more distantly related than when using the Splign-Compart (NCBI) option, but it is difficult to quantify how distantly related they can be. It should be noted that, when dealing with gene families, or genes that encode proteins with common protein motifs, partial annotations are usually obtained for many genes, although researchers are usually interested in full annotations only. The resulting CDS annotation is based on the homology to a given protein reference sequence, and thus may produce sequence annotations with lengths that are not multiple of three, if for instance, sequencing errors causing frameshifts are present in the genome to be annotated. Nevertheless, the existence of intron splicing signals at the exons 5’ and 3’ ends is taken into account. There will be no stop codon in the CDS annotation since the reference sequence is a protein. Splign-Compart (NCBI) runs considerably faster than ProSplign-Compart (NCBI), and thus users are advised to always try the Splign-Compart (NCBI) option first.

Here we attempt to obtain the Nicotiana attenuata PPCK1a CDS, using the Solanum tuberosum PPCK1a protein sequence (AF531415) as the reference.

1. Download the Solanum tuberosum PPCK1a protein sequence (AF531415) from here.
2. Import the AF531415.fasta file to BDBM using the File → Import FASTA operation (choose type Protein) or copy the file to the /fasta/proteins folder of the repository.
3. Download the Nicotiana attenuata genome assembly (GCA_001879085.1) from here and uncompress it in your computer.
4. Import the GCF_001879085.1_NIATTr2_genomic.fna file to BDBM using the File → Import FASTA operation (choose type Nucleotide) or copy the file to the /fasta/nucleotides folder of the repository.
5. Launch the Operations → ProSplign/ProCompart (NCBI) operation and choose the GCF_001879085.1_NIATTr2_genomic.fna file as Nucleotides FASTA (the genome file), the AF531415.fasta as Query protein FASTA (the file with the Solanum tuberosum PPCK1a protein sequence) and a file output name

A large file with many partial annotations will be produced but only three full-length CDS sequences (822, 831 and 834 bp long) will be obtained, that can be identified as those long sequences that start with an ATG (start codon). If only the start or end of the CDS is missing users may try to obtain the full sequence by using the BDBM “Refine annotation” option, which is not needed for this example. The main results file is saved in the /fasta/nucleotides folder that is located in the specified repository folder. The first number on the header is an index that is followed by the name of the protein sequence used to obtain the annotation. The remaining information gives the possibility to link this file to two other files that will be saved in the /Export Files/nucleotides folder that is located in the specified repository folder (see below), and information on the name of the sequence that was annotated (see text after Header:). In the /Export Files/nucleotides folder, the file with the txt extension shows the output of the tblastx search used for the subsequent annotation, while the file with the fasta extension gives the genome region where the gene has been annotated, including the name of the target nucleotide sequence (see text after Header:). The correspondence between the two files is made by looking at the first four numbers in the file with the Fasta extension that must match the first, second, fourth and fifth number, respectively, in the file with the txt extension. It should be noted that a single reference sequence can give rise to more than one annotation if ProSplign cannot completely confidently align the reference and target sequences (those positions that are confidently aligned are labelled with an asterisk in the file with the txt extension).