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.
/fasta/nucleotidesfolder of the repository) and inspected (double click on the file under the folder
These are basic operations but when dealing with large files, such as the one here considered, it may represent a major hurdle for someone without training in informatics.
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.
/fasta/nucleotidesfolder of the repository).
FraVesGETORF, the name used in this example). The output ORFs file will appear in the
FASTA filesfolder of the repository tree.
FraVesGETORFfile and by choosing the Make Blast Database operation. Choose a name for this nucleotide database, such as
/fasta/proteinsfolder of the repository).
tblastnsearch 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
It should be noted that if the goal is to produce a FASTA file with SFBB nucleotide sequences from several species, then the Operations → Get ORF operation should be performed separately for each species, and the appropriate prefix added (using the Operations → Reformat FASTA operation) to each file so that we can track the species of origin for each sequence. BLAST databases should be created separately for each file, and an alias created (using the Operations → BLAST DB Alias operation), so that all databases of interest will be treated as a single one. tblastn searches can then be performed using the SFBB protein sequence of interest. This will produce a FASTA file with the SFBB nucleotide sequences from all species considered. Please note that when using the Operations → Get ORF operation erroneous CDS annotations can be obtained due to the presence of sequencing gaps, or errors in the original genome/transcriptome sequences, so doing some checks on the sequences (after aligning them, for instance) is advisable.
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.
/fasta/nucleotidesfolder of the repository folder).
It should be noted that this operation can be done using multiple coding sequences at the same time, and that the header of the sequence used to produce a given annotation is transferred to the resulting FASTA file. Therefore, it is advisable to use the BDBM Operations → Reformat FASTA operation to produce a file that looks exactly as the researcher wants.
The Operations → Splign-Compart operation is not intended to be a gene annotation software, but rather a tool that allows the transference of a gene annotation available for a given species to another closely related species.
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).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.
/fasta/nucleotidesfolder of the repository).
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.
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
You can achieve this by using the Operations → Retrieve Search Entry operation
or your favourite text editor.
D_mel_sequences.fastafile using Operations → Make BLAST Database.
D_mel_sequences.fastafile 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.
D_mel_sequences.fastaFASTA file and use sequence name copied in the previous step.
Search Entrywill be created in BDBM with the contents of the selected sequence.
D_mel_sequences.fastaFASTA file using your favourite editor.
One advantage of the Operations → Splign-Compart operation is that it is insensitive to frameshifts caused by sequencing errors because the annotation is based on similarity at the nucleotide level and presence of putative splice sites only. Therefore, the resulting annotation should always be checked to see if the resulting CDS sequence lengths are multiple of three. It should also be noted that fast evolving genes are unlikely to be annotated this way.
As stated above, the Operations → Splign-Compart operation is not intended to be a genome annotation software but rather a useful tool that allows the easy retrieval of high quality CDSs to be used in detailed analyses. Using this approach we could get a complete CDS annotation similar to that of D. melanogaster for 303 CDSs.
For those cases where we could not get an annotation when using the D. melanogaster CDS, we tried to obtain an annotation based on our annotations for non-melanogaster species or when using trusted CDS sequences from FlyBase for non-melanogaster species. The former approach resulted in a modest increase in the number of CDS sequences (26 CDSs, 8% increase) that could be used, but the use of the FlyBase D. ananassae CDSs allowed the retrieval of D. bipectinata CDSs for 40% (17/42) of the genes here considered (see Annotations).
When the CDSs that are produced using the Operations → Splign-Compart operation are compared with those available at FlyBase, for 88 cases the CDS obtained with BDBM is identical to that available at FlyBase, for 58 cases the CDS obtained with the Operations → Splign-Compart operation is more similar to the D. melanogaster CDS than the one available at FlyBase (mainly due to errors in homopolymer runs that cause frameshifts, or to the use of a different translation start), and for two cases the CDS obtained with the Operations → Splign-Compart operation is less similar to the D. melanogaster CDS than the one available at FlyBase.
Therefore, overall, the Operations → Splign-Compart operation implemented in BDBM indeed produces CDS annotations similar to those available in FlyBase, and thus can be used to produce alternative CDS annotations when checking for suspicious gene annotations. For 40 non-melanogaster CDSs, we used the sequence from FlyBase, since the provided annotation is similar to the annotated D. melanogaster CDS. These sequences will be used in Use Case 6. The files with all gene annotations are available at here.
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.
/fasta/nucleotidesfolder of the repository).
UC2SFBB.fastafile and by choosing the option Make Blast Database.
tblastnsearch 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.
Exportsof the repository tree.
It is important to be careful when using custom additional parameters, as they can alter the format of the results, making them incompatible with other operations.
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.
/fasta/nucleotidesfolder of the repository or import them using the File → Import FASTA operation.
Ctrl + Aor
Cmd + Acombination and that you can select or unselect single sequences pressing
Cmdwhile clicking with the mouse.
Together with the options implemented in BDBM that allow the quick annotation of genes (see above), this means that it is possible to quickly produce multigene FASTA files for phylogenetic analyses.
For instance, we could use some of the files of Use Case 4 (those for which a gene annotation is obtained for all the species of interest) to prepare a file to be used in phylogenetic analyses of the Drosophila genus using a large variety of computer programs.
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.
/fasta/proteinsfolder of the repository.
/fasta/nucleotidesfolder of the repository.
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).