Codon, Initiator

RATT is programmed in ‘bash’ and ‘PERL’ and its design is illustrated in Figure 1 and Supplementary Figure S1. First, two sequences are compared using ‘nucmer’ from the MUMmer package (17 (link)) to define sequence regions that share synteny. Those regions are filtered using configurable parameters depending on the type of annotation mapping that is being attempted. Preset parameters are provided for transfers between assembly versions, strains or species (see Supplementary Table S1). To be included, the minimum nucleotide sequence identity between synteny blocks must be 40%. Synteny information is stored as a base range in the query and its associated base range in the reference. However, this information alone is inadequate to map the annotation because insertions or deletions (indels) change the relative distance between mapped synteny blocks. The coordinates are therefore sequentially adjusted across a synteny block by calling indels using ‘show-snp’ from the MUMmer package. Accurately calling indels within repetitive regions presents a particular challenge. Therefore, RATT recalibrates the adjusted coordinates using single nucleotide polymorphisms (SNPs, also called using ‘show-snp’) as unambiguous anchor points within synteny blocks. In transfers between very closely related sequences (e.g. successive assembly versions), SNPs may occur with insufficient frequency to perform this coordinate adjustment. In such cases, RATT modifies the query by inserting a faux SNP every 300 bp to aid in the recalibrating step. The final sequence and transferred annotations remain unaffected.
Figure 1.

Workflow of RATT.

Once the coordinates within synteny blocks have been defined, RATT proceeds to the annotation-mapping step, whereby each feature within a reference EMBL file is associated with new coordinates on the query (Supplementary Figure S1B). A feature is not mapped (and is put in the non-transferred bin file), if it bridges a synteny break and if its coordinate boundaries match different chromosomes, different DNA strands, or if the new mapped distance of its coordinates has increased by more than 20 kb. If a short sequence from the beginning, middle or the end of a feature can be placed within a synteny region, mapping is attempted (see Supplementary Figure S1B). In addition, if the exons of a single gene model map to different gene regions, the model is split and identified in the output file. The bin is an EMBL-format file that can be loaded onto the reference sequence for analysis (see Figure 2, brown colour track). Further outputs include statistics about transferred features or the amount of synteny conserved between the reference and query, as well as Artemis-readable files showing SNPs, indels and regions that lack synteny between the compared sequences, see the example on the sourceforge site.
Figure 2.

Transfer of annotation from the M. tuberculosis strain H37Rv onto the strain F11 sequence, over a deletion. The genomes of H37Rv (upper) and F11 (lower) are shown using the Artemis Comparison Tool (ACT). The source H37Rv annotation (light blue) is directly mapped onto F11 by RATT (green) except for those features corresponding to a region that is unique to the source strain that cannot be transferred and are written to a separate output file (brown).

Although two sequences may be related, differences can occur, such as a change in the start or stop codons of a protein-coding sequence. Therefore, we implemented a correction algorithm in RATT (see Supplementary Figure S1C). Figure 3 shows examples of the correction step. First, the start codon is checked. If it is not present, the upstream sequence is searched for a new start codon (Figure 3A). If a stop codon is found, the first start codon downstream is used. In the absence of any start codon, an error is recorded in the results file. If the sequence between exons has no stop codon and a length divisible by three bases but the splice acceptor or donor sequences are wrong, then the intron is eliminated. Likewise, frameshifts previously introduced into the reference to maintain conceptual translations (for instance, in apparent pseudogenes) will also be removed from coding sequences in the query. RATT will also detect, and attempt to fix, incorrect splice sites. As splice sites are difficult to annotate correctly, RATT only tries to correct a gene model that has one wrong splice site. If one incorrect splice site is detected, the closest alternative splice donor or acceptor is found that, when used, generates no frame shifts. Next, RATT searches for genes or exons with internal stop codons, further than 150 bp from the 3′-end. If the introduction of a frameshift would generate a model without internal stop codons, the model is corrected (Figure 3C). Stop codons are corrected last: if a model has less than five internal stops in its last exon, the model is shortened to the first stop codon (Figure 3B). If the model has no stop codon it is extended downstream until a stop codon is found.
Figure 3.

RATT corrections of transferred annotations. Annotation from H37Rv were transferred onto the F11 sequence (pale blue), corrected (green) and then compared with the existing strain F11 annotation in EMBL (yellow). (A and B) The correction of start and stop codons, respectively. In a more complex mapping situation (C), where all three reading frames are shown for clarity, RATT maps a large single coding sequence (CDS) from H37Rv to a locus within F11 that includes several in-frame stop codons. By inserting a frameshift (i.e. to indicate a pseudogene) the conceptual translation is preserved. This contrasts with two overlapping genes predicted as part of the F11 genome project.

Different criteria can be specified depending on the translation that an organism uses (e.g. such bacterial TTG and GTG start codons) or whether unsual splice sites are used. RATT is programmed in PERL and was tested in UNIX/LINUX environments. The output can be loaded into Artemis/Act. The list and explanation of all the output files can be found at the sourceforge site.

The crystal structure of selenium-binding protein (SeBP; PDB ID-2JZ7) was retrieved from
the RCSB Protein Data Bank (PDB) along with the gene and amino acid
sequence from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
The selenium-binding protein, SeBP, in Methanococcus
vannielii mediates selenium transport inside the cell.
During transport, SeBP sequesters selenium, regulating the amount
of free selenium in the cell, and delivers it specifically to the
selenophosphate synthase enzyme.^{22 (link)} SeBP
was a signal peptide-free protein confirmed by using the Signal P4.1
server (CBS, Denmark). Using a set of primers, the SeBP gene was amplified
by polymerase chain reaction (PCR) from the genomic DNA of M. vannielii. The reverse primer sequence 5′-CGGggtaccCCTACTCCGCGTCAAATGGTACCGC-3
and the forward primer sequence 5′-CGGggtaccTTCGAGGACAAATTCATTATCACCA-3
both introduce a restriction site KpnI on either
side of the start codon and after removing the stop codon, respectively.
DNA sequences of ECFP and Venus were amplified by PCR after shortening
the 5′ and 3′ ends to four codons and two codons each,
respectively. The ECFP and Venus fluorophores were attached to a bacterial
expression vector called pRSET-B (Invitrogen, USA) to create a cassette
(Figure S3). For a nanosensor construct
pRSET-B_ECFP_SeBP_Venus, inserting the amplified SeBP gene between
ECFP and Venus in the pRSET-B vector and fidelity of the construct
was confirmed by full-length sequencing (Figure S4). The pYES-DEST52 vector was used to carry the ECFP_SeBP_Venus
sequences by a gateway cloning method utilizing the LR-Clonase-II
enzyme (Invitrogen, USA) following the manufacturer’s instructions^{34 (link)} for expression of the sensor protein in yeast.
The S. cerevisiae/URA3 strain BY4742
was used for transforming the pYDEST-ECFP_SeBP_Venus plasmid as a
eukaryotic expression system. BY4742 strains were grown in liquid
YEPD (yeast extract peptone dextrose) agar medium at 30 °C with
aeration in a shaker. Following the manufacturer’s instructions
(Invitrogen, USA), competent cells of S. cerevisiae/URA3 were prepared and used for transforming with
the nanosensor sequences. The pcDNA3.1 (−) vector (Invitrogen)
was used to express the sensor protein in HEK-293T cells for mammalian
expression. By using the expression vector pRSET-B, the chimeric sequence
was cloned in the pcDNA3.1 (−) vector at the HindIII and BamHI sites.