LinearCDSfold is a tool for designing coding sequences by jointly optimizing their secondary structure stability and codon usage preferences.
Run the following command to compile LinearCDSfold.
make
To run LinearCDSfold, use the following command:
./LinearCDSfold [OPTIONS] <SEQUENCE_FILE>
SEQUENCE_FILE is an amino acid sequence file in FASTA format. The following is an example of SEQUENCE_FILE.
> P15421.fasta
MYGKIIFVLLLSGIVSISASSTTGVAMHTSTSSSVTKSYISSQTNGITLINWWAMARVIFEVMLVVVGMIILISYCIR
-c <CODON_USAGE_FILE>
CODON_USAGE_FILE is a CSV file containing codon usage frequencies, where each row specifies a codon triplet, the amino acid it encodes, and its relative usage frequency among synonymous codons.
Format of CODON_USAGE_FILE: [codon triplet], [amino acid], [relative frequency]
The following is an example of the content in CODON_USAGE_FILE. If the first line of CODON_USAGE_FILE starts with #, it is treated as a comment.
# triplet, amino acid, frequency
GCU,A,0.26
GCC,A,0.4
GCA,A,0.23
GCG,A,0.11
UGU,C,0.45
UGC,C,0.55
GAU,D,0.46
GAC,D,0.54
...
CODON_USAGE_FILE defaults to codon_usage_freq_table_human.csv.
-O <OBJECTIVE_FUNCTION>
The -O option (uppercase letter O) specifies the objective function for joint optimization of MFE (Minimum Free Energy) and CAI (Codon Adaptation Index). The OBJECTIVE_FUNCTION parameter must be set to either LD (default) or DN.
When OBJECTIVE_FUNCTION is LD, the objective function defined by LinearDesign is applied:
MFE − LAMBDA × l × log(CAI),
where l is the length of the input amino acid sequence, and LAMBDA is a real value ranging from 0 to ∞.
Conversely, if OBJECTIVE_FUNCTION is DN, the objective function defined by DERNA is utilized:
LAMBDA × MFE − (1 − LAMBDA) × l × log(CAI),
where LAMBDA is a real value ranging from 0 to 1.
-l <LAMBDA>
LAMBDA is a non-negative real-valued scaling parameter used to balance the contributions of the MFE of a coding sequence and its CAI value in the joint optimization objective.
When the objective function defined by LinearDesign is applied (i.e., OBJECTIVE_FUNCTION is set to LD), setting LAMBDA to 0 restricts the optimization to consider only MFE. Otherwise, both MFE and CAI are included, with higher values of LAMBDA increasing the weight assigned to CAI. If the LinearDesign objective function is used, the default value of LAMBDA is 0.
Conversely, when the objective function defined by DERNA is utilized (i.e., OBJECTIVE_FUNCTION is set to DN), setting LAMBDA to 1 restricts the optimization to consider only MFE. Otherwise, both MFE and CAI are included, with larger values of LAMBDA increasing the contribution of MFE. If the DERNA objective function is used, the default value of LAMBDA is 1.
-m <SEARCH_MODE> or --mode <SEARCH_MODE>
SEARCH_MODE is a string that selects the search strategy in LinearCDSfold. Available search modes are:
exact(default): Performs an exact search to obtain an optimal CDS.beam: Uses beam search to quickly generate an approximate CDS.pareto: Generates a set of Pareto-optimal CDSs using exact search and the DERNA objective function.
If no mode is specified, LinearCDSfold defaults to exact search mode.
-b <BEAM_SIZE>
BEAM_SIZE is a positive integer that specifies the beam size used by LinearCDSfold during beam search (i.e., -m beam is specified) and its default value is 500.
-t <TAU1> or --tau1 <TAU1>
TAU1 is a termination threshold used by LinearCDSfold when Pareto-optimal search is enabled (i.e., when -m pareto is specified). Its default value is 0.0025. In general, smaller values of TAU1 result in more Pareto-optimal CDSs being generated, at the cost of longer runtime.
-u <TAU2> or --tau2 <TAU2>
TAU2 is another termination threshold used by LinearCDSfold when Pareto-optimal search is enabled (i.e., when -m pareto is specified). Essentially, TAU2 is used to explore Pareto-optimal CDSs that are generated using LAMBDA values smaller than TAU1. Therefore, the value of TAU2 should be smaller than that of TAU1. By default, it is set to 0.00075. Smaller values of TAU2 allow more Pareto-optimal CDSs to be generated, but increase runtime.
-o <FILE_NAME>
The -o option (lowercase letter o) specifies the name of the output file, which will contain detailed results from LinearCDSfold in plain text format. The file FILE_NAME should use the .txt extension. If -o is not specified, the default output file is result.txt.
-f <FILE_NAME>
The -f option specifies the name of an additional output file that contains only the MFE and CAI results returned by LinearCDSfold for each LAMBDA value in CSV format. The FILE_NAME file should use the .csv extension. If -f is not specified, the default output file is result.csv.
> ./LinearCDSfold -l 2 -o P15421.txt example/P15421.fasta
Output: cat P15421.txt
Amino acid file: example/P15421.fasta
Codon usage table: codon_usage_freq_table_human.csv
Objective function: LinearDesign
Search mode: Exact search
Lambda: 2.000
Processing: [==================================================] 100%
Coding sequence and its secondary structure:
AUGUAUGGCAAGAUCAUCUUUGUGCUGCUGCUGAGCGGGAUCGUGUCGAUCUCCGCCAGCAGCACCACAGGGGUGGCCAUGCAUACCAGCACCAGCAGUAGCGUGACCAAGAGCUACAUCUCCAGCCAGACCAACGGCAUCACCUUGAUCAACUGGUGGGCCAUGGCCCGCGUGAUCUUCGAGGUGAUGCUGGUGGUGGUGGGGAUGAUCAUCUUGAUCAGCUACUGCAUCCGC
(((((((((.....((((((((((.((((((((.(((((((((...))))).)))))))))))).)))))))))))))))))))....((....((((((((.(((.(((((....((((((((.((..((((.((((((((((((((.......((((((....)))))).......)))))))))))))).))))))))))))))....))))).)))))))))))....))
Folding free energy: -132.600 kcal/mol
CAI: 0.919
Total runtime: 3.296 s
> ./LinearCDSfold -O DN -l 2 -o P15421_DN.txt example/P15421.fasta
Output: cat P15421_DN.txt
Amino acid file: example/P15421.fasta
Codon usage table: codon_usage_freq_table_human.csv
Objective function: DERNA
Search mode: Exact search
Lambda: 2.000
Processing: [==================================================] 100%
Coding sequence and its secondary structure:
AUGUAUGGCAAAAUCAUCUUCGUCUUGCUGCUCUCCGGGAUCGUAUCGAUCUCGGCGAGCAGCACGACGGGGGUGGCCAUGCAUACGAGUACGAGCAGUAGCGUGACUAAGAGUUAUAUAUCCUCACAGACCAACGGCAUCACCUUGAUAAAUUGGUGGGCGAUGGCCCGCGUAAUUUUCGAGGUGAUGCUGGUGGUCGUGGGGAUGAUAAUUCUUAUCAGCUACUGCAUUCGU
(((((((((.....((((((((((.((((((((.(((((((((...))))))))).)))))))).)))))))))))))))))))((((((....((((((((.(((.((((((((((.(((((((((.(((((.((((((((((((((.((((((((((((....)))))).)))))))))))))))))))).)))))))))))))))))))))))))))))))))))))))))
Folding free energy: -148.700 kcal/mol
CAI: 0.697
Total runtime: 3.310 s
> ./LinearCDSfold -m beam -b 100 -l 2 -o P15421_beam.txt example/P15421.fasta
Output: cat P15421_beam.txt
Amino acid file: example/P15421.fasta
Codon usage table: codon_usage_freq_table_human.csv
Objective function: LinearDesign
Search mode: Beam search
Beam size: 100
Lambda: 2.000
Processing: [==================================================] 100%
Coding sequence and its secondary structure:
AUGUAUGGCAAGAUCAUCUUUGUGCUGCUGCUGAGCGGAAUUGUGAGCAUUUCCGCCAGCAGCACCACAGGGGUGGCCAUGCAUACCAGCACCAGCAGUAGCGUGACCAAGAGCUACAUCUCCAGCCAGACCAACGGCAUCACCUUGAUCAAUUGGUGGGCCAUGGCCCGCGUGAUUUUCGAGGUGAUGCUGGUGGUGGUGGGGAUGAUCAUCUUGAUCAGCUACUGCAUCCGC
(((((((((.....((((((((((.((((((((.((((((.((....)).)))))))))))))).)))))))))))))))))))....((....((((((((.(((.(((((....((((((((.((..((((.((((((((((((((..((((.((((((....))))))..)))).)))))))))))))).))))))))))))))....))))).)))))))))))....))
Folding free energy: -130.300 kcal/mol
CAI: 0.924
Total runtime: 0.258 s
> ./LinearCDSfold -m beam -b 100 -O DN -l 0.001 -o P15421_beam_DN.txt example/P15421.fasta
Output: cat P15421_beam_DN.txt
Amino acid file: example/P15421.fasta
Codon usage table: codon_usage_freq_table_human.csv
Objective function: DERNA
Search mode: Beam search
Beam size: 100
Lambda: 0.001
Processing: [==================================================] 100%
Coding sequence and its secondary structure:
AUGUACGGCAAGAUCAUCUUCGUGCUGCUGCUGAGCGGCAUCGUGUCCAUCAGCGCCAGCAGCACCACCGGCGUGGCCAUGCACACCUCCACCAGCAGCAGCGUGACCAAGAGCUACAUCAGCUCUCAGACCAAUGGCAUCACCCUGAUCAACUGGUGGGCCAUGGCCAGGGUGAUCUUCGAGGUGAUGCUGGUGGUGGUGGGCAUGAUCAUCCUGAUCAGCUACUGCAUCAGG
..((((((..(((...)))))))))((((((...))))))((((((((((((.((((((((.((((.(((.(.((((((((......((((((((...(((.(((((((((((((.....))))))........)))..)))).))).....)))))))).)))))))).).))......).)))).)))))))).))))))))))))....(((((((((...))).))))))
Folding free energy: -103.100 kcal/mol
CAI: 0.991
Total runtime: 0.263 s
./LinearCDSfold -m pareto -o P15421_Pareto.txt -f P15421_Pareto.csv example/P15421.fasta
Output: cat P15421_Pareto.csv
lambda,MFE,CAI
0.000010,-88.000000,1.000000
0.999990,-148.700000,0.733673
0.500000,-148.700000,0.733673
0.250005,-148.700000,0.733673
0.125007,-148.700000,0.733673
0.062509,-148.300000,0.760887
0.031259,-148.300000,0.760887
0.093758,-148.700000,0.733673
0.015635,-146.100000,0.798641
0.078133,-148.700000,0.733673
0.007822,-137.700000,0.882926
0.023447,-148.300000,0.760887
0.070321,-148.700000,0.733673
0.003916,-128.400000,0.939532
0.011729,-146.100000,0.798641
0.019541,-148.300000,0.760887
0.066415,-148.700000,0.733673
0.001963,-124.300000,0.956380
0.005869,-134.700000,0.905781
0.009775,-143.400000,0.829243
0.017588,-147.500000,0.775186
0.064462,-148.300000,0.760887
0.000987,-103.100000,0.990816
0.000498,-97.200000,0.996524
0.001475,-118.800000,0.968451
Corresponding author: Prof. Chin Lung Lu (Email: cllu@cs.nthu.edu.tw)