RNAlign2D

RNAlign2D is a Python wrapper allowing one to align multiple RNA molecules using information about secondary structure and sequence. For this purpose MUSCLE program is used, but one can adjust it to use any other multiple sequence alignment tool.

This tool firstly remove common RNA modifications, then convert RNA sequences and secondary structures to pseudo-amino acid sequences (please not mistake for translation process - that is only technical solution), and use MUSCLE to align pseudo-amino acid sequences. Finally process is reverted and alignment with original sequence and secondary structure is restored.

Dot bracket format

Format, where paired residues as marked as brackets () and unpaired residues are marked as dots. For example simple structure containing hairpin (stem of 5 residues length, loop of 6 residues length) may look like: ........(((((......))))).........

For pseudoknots [] brackets are used. For higher level pseudoknots {}, <> or even Aa, Bb, Cc ... pairs are used. For the RNAlign2D maximum pseudoknots level taken into account is Bb.

Input and output files

Input and output files are modified FASTA format. For each sequence in the file: * First line contain > character and then optional name * Second line (or multiple lines) contain sequence * Third line (or multiple lines) contain structure Sequence and structure are distinguished on the basis of dot and bracket content. If there is no secondary structure, linear - unpaired structure is assumed. If there is Vienna RNA package installed, this package would automatically generate missing secondary structures.

Sequence may contain modified nucleotides - these modifications of nucleotide residues are removed (for example B - 2′-O-methylcytidine is converted to C - cytidine), and after the alignment they are restored back to the original state. RNAlign2D uses modifications abbreviations from MODOMICS database. Secondary structure may contain higher level pseudoknots, but for the simple mode only fist level - [] pseudoknots are treated differently, and for the pseudo mode only pseudoknots up to level Bb.

Modes

• Mode simple
• Mode pseudo

Mode simple is used in most cases, when there are no pseudoknots or there are only first level pseudoknots. It uses both sequence and secondary structure into account. Higher level pseudoknots are treated as unpaired regions. Mode pseudo on the other hand uses only secondary structure, as it allows to align higher level pseudoknots. In some cases mode simple may give better results than mode pseudo even for sequences. In case of the mode pseudo there is a need to explicitly show matrix dedicated for pseudo alignment, otherwise results may not reflect anything.

Options

• -i option - input file
• -o option - output file
• -matrix option - file with custom matrix or default matrix for pseudoknots (data/pseudo_matrix in the project)
• -mode option - allow to select one of the modes: simple or pseudo, default simple, please remember to use proper matrix for given mode
• -gapopen option - allow to select gap opening penalty for sequence alignment
• -gapextend option - allow to select gap extending penalty for sequence alignment

example usage:

rnalign2d -i my_input -o my_output

Custom matrix

One can define matrix for himself. As it is simply text file, there is nothing that cannot be done manually. Hovewer there is script create_matrix, that can facilitate that process.

• Most probably the strongest score should be given to the same type of brackets like ( and ( or [ and [, default value is 7, option: -same
• Probably the smallest score should be given to the oposite brackets like ( and ), default value is -10, option -reverse
• Sometimes there is a bulge in the RNA structure or the loop is bigger by one pair of residues, therefore situation like ( and . should be given small negative value, default value is -1, option -bracket-dot
• In the situation that in one structure there is pseudoknot and in the other there is classical Watson-Crick base pair for example ( and [, there should be given small positive or negative score, default value is 2, option -other
• In case of two unpaired regions . and . there should be positive score, but we suggest it to be small, as loops are probably the best place to introduce gaps, default value is 3, option -dot_dot
• For the simple mode there is also possibility to add score for matching nucleotide residues in the sequence. Depending on how important the sequence is, this value may vary, default value is 1, option -seq_match_add
• In case of pseudo mode you need to define mode (options are simple and pseudo), default value is simple, option -mode
• Output name could be defined using -o option

exaple usage:

create_matrix -o my_result -seq_match_add 8

Remove modifications

One can remove modifications from file for this there is script rm_mod There are options -i to define input file and -o to define output file

exaple usage:

rm_mod -o my_result -i my_input

REQUIREMENTS

• Python 3 (tested on Python 3.5)
• MUSCLE (tested on version 3.8.31)
• pytest (tested on version 5.1.3)
• Vienna RNA (optional, tested on version 2.4.14)

EXAMPLE FILES

In the directory example there are 3 files:

• dot_bracket.txt - file containing example input sequence
• dot_bracket2.txt - file containing example output
• dot_bracket_cls.txt - file containing only sequences

Files are created using data from T-psi-C-database_tpsic.igcz.poznan.pl

OTHER

In case of WARNING: *** WARNING *** Matrix is not symmetrical, �->�=-10, �->�=0 most probably there is no problem at all, but if it is your custom matrix, you can check it to ensure it contain desired data

To test this software Vienna RNA is required, otherwise one test would fail.

CITATION

If you are using our software in your research - cite us: <manuscript in preparation>