Table 7.

Fold-then-align RSP tools based on comparative sequence analysis

Characteristic			Fold-then-align RSP Tool	Description	Input	Output	Applicable Species	Active (T)/Inactive (F)
Conservation	Suboptimal Prediction	Local Interaction Length	MARNA (surpassed by LOcARNA) [252, 253]	An MSA method that considers both primary sequence and secondary structure, and is based on pairwise comparison with edit operations on arcs and bases	RNA sequences in FASTA format (max 3 for RNAsubopt)	Prediction of the consensus sequence and structure Structure computation via MFE (RNAfold); structural shape (RNAshapes); structural ensemble (RNAsubopt) Computation speed faster than MASTR	Eukaryote	F
			planACstar (RNA–RNA) [196]	A tool for fine-tuning the folding process and structural RNA alignments in the twilight zone	A set of alignments with several sequences per alignment	Prediction of conserved RNA secondary structure and offer improvement in the twilight zone via a combination of several tools: ClustalW, RNAalifold, RNAfold, RNAforester, and RNAalifold	Mammal	T
			RNAspa (part of ViennaRNA package) (ncRNA) [270]	A shortest path approach for comparative prediction of the secondary structure of ncRNA molecules via a simple string Edit-Distance algorithm	A set of unaligned RNA sequences	Prediction of the secondary structure for a set of ncRNAs in linear time in the number of molecules Generation of graph, where the layer of vertices represents the suboptimal solutions	Virus, bacteria	T
			RNAcast (RNA consensus abstract shape technique) (ncRNA) [193]	An alternative to the Sankoff algorithm for multiple RNA structure prediction	At least 2 RNA sequences	Enumeration of the near-optimal abstract shape space Prediction of the consensus of an abstract shape common to all sequences Prediction of the thermodynamically best structure with the common shape for each sequence Prediction of the consensus structures of ten or more sequences at once	Virus	F
	No Suboptimal		RNA Sampler (ncRNA-RNA) [198]	A sampling-based algorithm for common secondary RSP and structural alignment via graph-theoretical approach, with no limitation on predicting pseudoknots; and provide refinement of alignment and folding process	Two RNA sequences	Prediction of common RNA secondary structures in multiple unaligned sequences Measurement of stem conservation by adopting the stem assembly idea from comRNA [271]; and combining both intrasequence base pairing and intersequence base alignment probabilities	Animal, eukaryote	T
Conservation	No Suboptimal	Global interaction length	MASTR (multiple alignment and structure prediction of ncRNAs) [197]	A tool to solve simultaneous structure prediction and MSA, while providing refinement of alignment and folding process	At least 2 RNA sequences in FASTA format	Prediction of the consensus structures Possibility to add structural constraints Computation speed faster than FoldalignM	Human, eukaryote	T
			LaRA 2 (ncRNA-RNA) [200, 272]	A parallel and vectorised program for sequence-structure alignment of RNA sequences and capable of handling arbitrary pseudoknots	At least 2 RNA sequences in FASTA format	Analysis of large sets of RNA secondary structures in a relatively short time, based on structural alignment Derivation of structural motifs (based on the produced alignments) to search in genomic databases	Bacteria, virus, eukaryote	T
			T-Coffee (tree-based consistency objective function for alignment evaluation) [195]	A web server for the RNA MSA using structural information and homology extension	RNA, DNA and protein alignments from any source in FASTA format	Combination of a collection of multiple or pairwise; global or local alignments into a single tool Estimation of the level of consistency/alignment accuracy of each position within the new alignment with the rest of the alignments Evaluation of RNA alignment and outputs a coloured version indicating the local reliability Evaluation of MSA using structural information with APDB and iRMSD Other types of T-coffee-related tools:   a)M-Coffee- Alignment of RNA by combining the output of popular aligners b)R-Coffee- Alignment of RNA sequences using predicted secondary structures c)SARA-Coffee- Alignment of RNA sequences using tertiary structure	Parasite, bacteria, animal	T
	Suboptimal Prediction		CMfinder (ncRNA) [199]	A highly accurate covariance model-based RNA motif finding tool, derived from a small number of related sequences, to identify homologues in deeply diverged species	Unaligned RNA sequences	Prediction of RNA motif Inference of alignment and consensus secondary structure of an RNA Indication of the evidence of RNA secondary structure within the alignment. Summary of tools: RNAphylo: Assignment of a probabilistic score to an existing alignment, using an explicit phylogenetic model Hmmpair: Assignment of a score based on evidence of covariation that is supported by sequence conservation ScoreMotif.pl script: Combination of the previous two scores into one	Bacteria, archaea	T
			RNAforester (part of ViennaRNA package) [273]	A software comparing RNA secondary structures via forest alignment	RNA secondary structures from stdin or RNA sequences and structures in FASTA format	Calculation and comparing pairwise and multiple RNA secondary structure alignments via the tree alignment model Generation of alignments in ASCII format written to stdout Postscript drawings of structure alignments via option -2D	Bacteria, virus, eukaryote	T

2D: 2-dimensional; ASCII: American standard code for information interchange; DNA: deoxyribonucleic acid; MSA: multiple sequence alignment; ncRNA: noncoding RNA; RNA: ribonucleic acid; T-Coffee: tree-based consistency objective function for alignment evaluation