Team:StanfordBrownSpelman/Modelling

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Revision as of 19:34, 6 October 2014

DoubleOptimizer: a utility for simultaneous codon optimization and optimization for gene synthesis

Background

Gene synthesis as a tool for biological engineering presents both opportunities and challenges. One opportunity presented is the ability to optimize codon usage in a gene to match that of a host organism. Compared to traditional cloning methods, this can increase protein yields in the host organism by several fold. However, while there exist a large number of freely-usable programs that perform codon optimization, there is no guarantee that the sequences these programs provide will be able to be synthesized. Specifically, in the case of genes with repetitive amino acid sequences, these programs will often generate outputs that contain too many repeated short DNA sequences to be synthesized commercially.

As an example, the hypothetical protein X777_06170 from the ant species Cerapachys biroi has an amino acid sequence that appears to be somewhat repetitive:

       1 mklfkclvpv vvlllikdss arpglirdfv ggtvgsilep fqilkpkdsy adanshasah
      61 nlggtfslgp vslggglssa sasssasang gglasasska daqaggygyg gsnanaqasa
     121 sanaqgggyg nggihgiypg qqgvhggnpf lggagsnana naiananaqa naggnngglg
     181 syggyqqggn ypidsstgpi gnnpflsggh gdgnanaaan anagasaign gggpidvnnp
     241 flhggaansg agginyqpgn aggiilsekp lglptiypgq hppayldsig spgansnaga
     301 napcsecgss gatilgyegq glggikesgs sgatilgyeg qglggikesg ssgatilgye
     361 gqglggikes gssgatilgs ydgqgpsgat ilgdyngqgl ggikessgvt vlgdyegqgl
     421 ggisgphggh gqaganagan ananagatvg ssggvlggvg dhggyhgyng hdgssglnlg
     481 gygggsnana qassnalass ggsssatsda lsnahssggs alanssskas angsgsanan
     541 ahassnassg shglgsktsa ssqasasadt rdmlifs

Note that this sequence is not simply the same sequence repeated multiple times, but instead contains several motifs on the order of 10 - 20 amino acids in length that occur several times. When this sequence was run through the codon optimization program for expression in E. coli provided by a major DNA synthesis firm, the resulting output could not be synthesized by the very same firm: the "optimized" DNA sequence contained too many recurring short (> 8 nucleotide) DNA sequences to allow for synthesis.

Manually correcting for repeats in the codon-optimized DNA sequence is a sub-optimal solution: not only is this process time-consuming, but it has the tendency to undo the codon-optimization: if a sequence of amino acids occurs several times, one may be forced to use all possible codon-combinations to represent this sequence to avoid nucleotide-sequence repetition. Unless corrected for by skewing codon usage elsewhere in the sequence, this will tend to make the codon usage more uniform than is optimal for the expression vector. Additionally, any changes made in either correcting for repeats or re-correcting for codon usage may in turn introduce additional repeats.

Solution: DoubleOptimizer

DoubleOptimizer is a software tool we have created to optimize codon usage in a gene both to match a given codon usage distribution and to avoid repetition of nucleotide sequences. Given a DNA or amino acid sequence and a desired codon distribution, DoubleOptimizer will produce, within a matter of minutes, an equivalent sequence that has substantially reduced DNA sequence repetition, while also closely matching the desired codon usage.

Availability and Usage

DoubleOptimizer may be downloaded here. (Note: this link is not public yet, but it will be converted to public before Wiki Freeze Day.)

DoubleOptimizer is a command line utility, provided as a Java jar file. It can be invoked from command line on any system with Java installed, with the following syntax:

      java -jar DoubleOptimizer.jar seq.txt codons.txt [Optional flags]

where "seq.txt" is a DNA sequence, stored as a plain text file, and "codons.txt" is a file containing the desired codon distribution to match. It should be formatted as plain text, according to the following example template:

      GCG .36
      GCC .27
      GCA .21
      GCT .16
      R
      CGC .40
      CGT .38
      CGG .10
      CGA .06
      AGA .04
      AGG .02
      N
      AAC .55
      AAT .45
      D
      GAT .63
      GAC .37
      C
      TGC .55
      TGT .45
      E
      GAA .69
      GAG .31
      Q
      CAG .65
      CAA .35
      G
      GGC .40
      GGT .34
      GGG .15
      GGA .11
      H
      CAT .57
      CAC .43
      I
      ATT .51
      ATC .42
      ATA .07
      L
      CTG .50
      TTG .13
      TTA .13
      CTT .10
      CTC .10
      CTA .04
      K
      AAA .77
      AAG .23
      M
      ATG 1
      F
      TTT .57
      TTC .43
      P
      CCG .52
      CCA .19
      CCT .16
      CCC .12
      S
      AGC .28
      AGT .15
      TCG .15
      TCT .15
      TCC .15
      TCA .12
      *
      TAA .64
      TGA .29
      TAG .07
      T
      ACC .44
      ACG .27
      ACT .17
      ACA .13
      W
      TGG 1
      Y
      TAT .57
      TAC .43
      V
      GTG .37
      GTT .26
      GTC .22
      GTA .15

(Eli, can you help me make this collapsible??)

(Note that the above example is actually the codon usage distribution of E. coli.)

DoubleOptimizer supports non-canonical codon assignments: the amino acid-codon groupings can by specified in whatever way the user wants in the codon distribution file.

When executed, DoubleOptimizer will first display the input sequence with repetitive regions highlighted. It will also give the fraction of the sequence that initially consists of repetitive regions (defined by default as regions of 8 nucleotides or more that occur more than once in the sequence, including as their reverse complement), and a chi-squared value for the goodness-of-fit to the desired codon distribution (lower is better).

DoubleOptimizer will then compute and display the optimized sequence (By default, it will produce the best sequence it can find after 10 seconds of computation time). Again, repetitive regions will be highlighted, and the same measurements of repetitiveness and codon fit will be given.

The following optional flags may be used to change the program's behavior:

• -A

This allows for an amino-acid sequence, specified in single-letter code, to be used as input instead of a DNA sequence. The initial sequence statistics displayed will be for a uniform random reverse translation of the given amino acid sequence.

Example:

java -jar DoubleOptimizer.jar aaseq.txt codons.txt -A

• -T##

This allows the user to specify, in seconds, a different run-time for the program other than the default 10 seconds. While 10 seconds should be sufficient to produce a well-optimized result for most genes of moderate length on modern desktop computers, longer times may produce better-optimized results on slower machines or on longer sequences.

Example:

java -jar DoubleOptimizer.jar aaseq.txt codons.txt -A -T30

• -L##

This allows the user to specify a different minimum length for what is considered a repeat, other than the default 8 nucleotides.

Example:

java -jar DoubleOptimizer.jar seq.txt codons.txt -L7 -T15

• -S##, -E##

These allow the user to specify the starting and ending nucleotide, respectively, of the coding region in a construct sequence to be sythesized. Nucleotides outside this frame will be ignored for codon usage optimization, and will never be modified. This option is useful for preventing repetitions, within the coding region, of fixed sequences that occur at the ends of a construct to be sythesized, outside of the coding region. The default values are the beginning of the sequence, and the end of the last complete codon. These options may be used together or independently. Values are one-indexed. If used with -A, these will be interpreted as amino acid indices.

Example:

java -jar DoubleOptimizer.jar seq.test ecoli2.txt -S121 -E1853 -R1000 -D100

• -D##

This option will make the program periodically display the current best sequence, and associated statistics, as it runs. The number given is the number of optoimization cycles the program will perform between each round of displaying the sequence. This provides a continuous measure of progress on long optimization runs. Note that, when given 10 seconds, the program may execute several thousand cycles of optimization, so an argument on the order of 100 may be reasonable.

Example:

java -jar DoubleOptimizer.jar seq.test ecoli2.txt -S121 -E1853 -R1000 -D100

• -R##

This option will make the program halt optimization after a certain number of optimization cycles. This may be used with a large value of -T to standardize optimization quality between computers of different speeds. Because this option is mostly only useful for testing the efficiency of this program itself, it may be removed from future releases.

Example:

java -jar DoubleOptimizer.jar seq.test ecoli2.txt -S121 -E1853 -T1000 -R1000

Case Studies and Performance Data

• To be added

References

• To be added

CompositionSearch: a simple utility for local, fast search of protein sequences for matches to a specified amino acid distribution.

Background

When the relative proportions of amino acids in an unknown protein product have been chemically determined, it is often useful to search a proteome for proteins that have similar amino acid distributions, in order to identify this protein product. While at least one online utility for performing this task already exists (provided by the Swiss Institute of Bioinformatics, http://web.expasy.org/aacompident/aacompfree.html here), the web-based nature of this program creates some limitations. Firstly, the SIB provides computational resources for the calculation, resulting in slower turnaround for the user (searches take about 15 minutes). Secondly, this program will only search for proteins already in the Swiss-Prot / TrEMBL databases. (At last check, only the smaller Swiss-Prot search was functional.) If an organism is being newly studied and has just been sequenced, its predicted proteome will not be in these databases. Thirdly, due to limited resourses, only the top matches to a given search are provided. This does not allow for statistical comparison to the "typical" protein within a given proteome. Fourthly, for very-high-security tasks, uploading data to a third party may be undesirable.

Solution: CompositionSearch

CompositionSearch is a software tool we have created to address these issues by allowing an individual to rank all proteins in a proteome by similarity (minimum Euclidian distance) to a reference amino acid distribution locally on one's own computer. This ranking can be generated in a matter of seconds, rather than taking several minutes. Because it ranks all proteins in a proteome, CompositionSearch can also generate a figure for the significance of the similarity of a given protein to a given amino acid distribution, using the similarity of the rest of the proteome as a statistical distribution function.

Availability and Usage CompositionSearch may be downloaded here. (Note: this link is not public yet, but it will be converted to public before Wiki Freeze Day.)

DoubleOptimizer is a command line utility, provided as a Java jar file. It can be invoked from command line on any system with Java installed, with the following syntax:

     java -jar CompositionSearch.jar prot.fasta freqs.txt out.csv [Optional flags]

where "prot.fasta" is a FASTA-formated proteome, and "freqs.txt" is a file containing the desired amino distribution to match. It should be formatted as plain text, according to the following example template:

A 0.134	
D 0.044	
E 0.04	
G 0.228	
I 0.033	
K 0.021	
L 0.04	
P 0.08	
R 0.009	
S 0.151	
T 0.038	
V 0.083

(Eli, can you help me make this collapsible??)

Note that this may contain as many or as few amino acids as desired. The frequences, however, are interpreted as absolute, so if all amino acids are represented, they should add 1. (See below for discussion of how the optional -N and -X flags affect this interpretation.)

"out.csv" in the above example line is the destination path to store the results of the calculation. The output will be a spreadsheet in csv format, which may be imported into your favorite desktop spreadsheet application (i.e. Microsoft Excel, LibreOffice Calc, etc.). After execution, out.csv will contain a spreadsheet showing the reference distribution, and, in order by similarity to the reference distribution, the amino acid distributions of all proteins in the proteome. Protein name, similarity ranking, similarity score (Euclidian distance between amino acid distributions; lower is better), and similarity p-value (on the curve of other proteins in rthe proteome) will be listed for each protein.