Sequence File Formats:  FASTA and SEQ

Nucleotide Sequences can be provided to RNAstructure in either FASTA or SEQ format.

In FASTA files, each nucleotide sequence begins with a single-line description that must start with the greater-than symbol (>). Subsequent lines should only contain the sequence itself.  The sequence may be formatted with whitespace, which is ignored, however blank lines are not allowed in the middle of FASTA input. FASTA files should have a ".fasta" extension.

>Title of Sequence
AAA GCGG UUTGTT UTCUTaaTCTXXXXUCAGG
UUA GCCG UUTGTT UTCUTaaTCTGGG

SEQ files have the following format:

1. Comment lines must be at the beginning of the file and must each start with a semicolon.  At least one comment line is required. Additional comment lines are allowed as long as each starts with a semicolon.
2. The title of the sequence must be provided on a single line immediately following the comment line(s).
3. The sequence must start on the line after the title. It should be entered from 5' to 3' and can include spaces and line breaks for formatting.
4. Finally, the sequence must end in "1" (the character representing the number one).

; Comments must start with a semicolon.
; There can be any number of comments, but at least one is required.
A sngle-line title must immediately follow the comment(s).
AAA GCGG UUTGTT UTCUTaaTCTXXXXUCAGG1

Important notes regarding sequences in RNAstructure:

• Nucleotide sequences can contain U or T interchangably. These will be interpreted based on the context of the desired operation (i.e. as U in RNA calculations or as T in DNA calculations). 
• Spaces are allowed in the sequence, and will simply be ignored.
• Sequences are case-sensitive and should generally be in CAPITAL letters. Lowercase letters indicate a base that should be forced  single-stranded (unpaired) in the predicted structure. 
• "XXXX" can be used in the sequence to indicate that some bases have been left out of the prediction.

CT File Format

A CT (Connectivity Table) file contains secondary structure information for a sequence. These files are saved with a CT extension. When entering a structure to calculate the free energy, the following format must be followed.

1. Start of first line: number of bases in the sequence
2. End of first line: title of the structure
3. Each of the following lines provides information about a given base in the sequence. Each base has its own line, with these elements in order:
   • Base number: index n
   • Base (A, C, G, T, U, X)
   • Index n-1
   • Index n+1
   • Number of the base to which n is paired. No pairing is indicated by 0 (zero).
   • Natural numbering. RNAstructure ignores the actual value given in natural numbering, so it is easiest to repeat n here.

The CT file may hold multiple structures for a single sequence. This is done by repeating the format for each structure without any blank lines between structures.

The CT file format is such that any files generated by RNAstructure are compatible with mfold/Unafold (available from Michael Zuker), and many other software packages.

300 ENERGY = 7.0 example
1 G 0 2 22 1
2 G 1 3 21 2
3 G 2 4 20 3
4 C 3 5 19 4
5 G 4 6 0 5
6 A 5 7 0 6
7 A 6 8 0 7
8 U 7 9 18 8
9 U 8 10 17 9
10 G 9 11 16 10
(structure continues to next structure...)
300 ENERGY = 6.2 example
1 G 0 2 0 1
2 G 1 3 0 2
3 G 2 4 20 3
4 C 3 5 19 4
5 G 4 6 0 5
6 A 5 7 0 6
7 A 6 8 0 7
8 U 7 9 18 8
9 U 8 10 17 9
10 G 9 11 16 10
(structure continues to next structure or end of file...)

Constraint File Format

Folding constraints are saved in plain text with a CON extension. These can be hand edited. For multiple entries of a specific type of constraint, entries are each listed on a separate line. When there is no constraint of a type, there are no lines required. Note that all specifiers, followed by "-1" or "-1 -1", are expected by RNAstructure. For all specifiers that take two arguments, it is assumed that the first argument is the lower base pair number. The file format is as follows:

DS:
XA
-1
SS:
XB
-1
Mod:
XC
-1
Pairs:
XD1 XD2
-1 -1
FMN:
XE
-1
Forbids:
XF1 XF2
-1 -1

• XA: Nucleotides that will be double-stranded
• XB: Nucleotides that will be single-stranded (unpaired)
• XC: Nucleotides accessible to chemical modification
• XD1, XD2: Forced base pairs
• XE: Nucleotides accessible to FMN cleavage
• XF1, XF2: Prohibited base pairs

DS:
15
25
76
-1
SS:
17
18
20
35
-1
Mod:
2
15
-1
Pairs:
16 26
-1 -1
FMN:
-1
Forbids:
15 27
-1 -1

SHAPE Reactivity File Format

The file format for SHAPE reactivity comprises two columns. The first column is the nucleotide number, and the second is the reactivity.

Nucleotides for which there is no SHAPE data can either be left out of the file, or the reactivity can be entered as less than -500. Columns are separated by any white space.

Note that there is no header information. Nucleotides 1 through 10 have no reactivity information. Nucleotide 11 has a normalized SHAPE reactivity of 0.042816. Nucleotide 12 has a normalized SHAPE reactivity of 0, which is NOT the same as having no reactivity when using the pseudo-energy constraints.

By default, RNAstructure looks for SHAPE data files to have the file extension SHAPE, but any plain text file can be read.

9 -999
10 -999
11 0.042816
12 0
13 0.15027
14 0.16201

List File Format

List files have a LIS extension. This file contains any number of sequences of any length or nucleic acid, each on its own line.

CUGAGCCAAG
GGGCUCAACG
GGCGUGAGAAAC

Alignment File Format

Alignment files are plain text. They are formatted as a nucleotide in the first sequence immediately followed by the nucleotide in the second sequence it's aligned to, separated by a space. Only one alignment pair can be on each line, and the last line of the file must be "-1 -1".

10 12
11 13
-1 -1