nuad 0.4.6

nuad stands for "NUcleic Acid Designer". Enables one to specify constraints on a DNA (or RNA) nanostructure made from synthetic DNA/RNA and then attempts to find concrete DNA sequences that satisfy the constraints.

nuad

nuad is a Python library that enables one to specify constraints on a DNA (or RNA) nanostructure made from synthetic DNA/RNA and then attempts to find concrete DNA sequences that satisfy the constraints.

Note: If you are reading this on the PyPI website, many links below won't work. They are relative links intended to be read on the GitHub README page.

Table of contents

• Overview
• API documentation
• Installation
  • Installing nuad
  • Installing NUPACK and ViennaRNA
• Data model
• Constraint evaluations must be pure functions of their inputs
• Examples
• Parallelism
• Reporting issues
• Contributing
• NUPACK Copyright Notice

Overview

nuad stands for "NUcleic Acid Designer".† It is a Python library that enables one to specify constraints on a DNA (or RNA) nanostructure made from synthetic DNA/RNA (for example, "all strands should have complex free energy at least -2.0 kcal/mol according to NUPACK", or "every binding domain should have binding energy with its perfect complement between -8.0 kcal/mol and -9.0 kcal/mol in the nearest-neighbor energy model"), and then attempts to find concrete DNA sequences that satisfy the constraints. It is not a standalone program, unlike other DNA sequence designers such as NUPACK. Instead, it attempts to be more expressive than existing DNA sequence designers, at the cost of being less simple to use. The nuad library helps you to write your own DNA sequence designer, in case existing designers cannot capture the particular constraints of your project.

Note: The nuad package was originally called dsd (DNA sequence designer), so you may see some old references to this name for the package.

†A secondary reason for the name of the package is that some work was done when the primary author was on sabbatical in Maynooth, Ireland, whose original Irish name is Maigh Nuad.

API documentation

The API documentation is on readthedocs: https://nuad.readthedocs.io/

Installation

nuad requires Python version 3.7 or higher. Currently, although it can be installed using pip by typing pip install nuad, it depends on two pieces of software that are not installed automatically by pip (see issue #12).

nuad uses NUPACK and ViennaRNA, which must be installed separately (see below for link to installation instructions). While it is technically possible to use nuad without them, most of the pre-packaged constraints require them.

To use NUPACK on Windows, you must use Windows Subsystem for Linux (WSL), which essentially installs a command-line-only Linux inside of your Windows system, which has access to your Windows file system. If you are using Windows, you can then run python code calling the nuad library from WSL (which will appear to the Python virtual machine as though it is running on Linux). WSL is necessary to use any of the constraints that use NUPACK 4.

Installing nuad

To install nuad, you can either install it using pip (the slightly simpler option) or git. No matter which method you choose, you must also install NUPACK and ViennaRNA separately (see instructions below).

• pip

  At the command line (WSL for Windows, not the Powershell prompt), type

  pip install nuad
  

• git

  This method has more steps, but it might be preferable if you want to use a new feature that is not on the main branch: the package installed by the pip instructions above will install the version currently on the main branch.

  1. Download the git repo, by one of two methods:

     • Install git if necessary, then type

       git clone https://github.com/UC-Davis-molecular-computing/nuad.git

       at the command line, or

     • on the page https://github.com/UC-Davis-molecular-computing/nuad, click on Code → Download Zip:

       

       and then unzip somewhere on your file system.

  2. Add the directory nuad that you just created to your PYTHONPATH environment variable. In Linux, Mac, or Windows Subsystem for Linux (WSL), this is done by adding this line to your startup script (e.g., ~/.bashrc, or ~/.bash_profile for Mac OS), where /path/to/nuad represents the path to the nuad directory:

     export PYTHONPATH="${PYTHONPATH}:/path/to/nuad"
     

  3. Install the Python packages dependencies listed in the file requirements.txt by typing

     pip install numpy ordered_set psutil pathos xlwt xlrd tabulate scadnano
     

     at the command line. If you have Python 3.7 then you will also have to install the typing_extensions package: pip install typing_extensions

Installing NUPACK and ViennaRNA

Recall that if you are using Windows, you must do all installation through WSL (Windows subsystem for Linux).

Install NUPACK (version 4) and ViennaRNA following their installation instructions (NUPACK installation, ViennaRNA installation, and ViennaRNA downloads). If you do not install one of them, you can still install nuad, but most of the useful functions specifying pre-packaged constraints will be unavailable to call.

After installing ViennaRNA, it may be necessary to add its executables directory (the directory containing executable programs such as RNAduplex) to your PATH environment variable. (Similarly to how the PYTHONPATH variable is adjusted above.) NUPACK 4 does not come with an executable, so this step is unnecessary; it is called directly from within Python.

Type python at the command line, then type import nuad. It should import without errors:

$ python
Python 3.7.5 (default, Nov  7 2019, 10:50:52)
[GCC 8.3.0] on linux
Type "help", "copyright", "credits" or "license" for more information.
>>> import nuad
>>>

To test that NUPACK and ViennaRNA can each be called from within the Python library (note that if you do not install NUPACK and/or ViennaRNA, then this will fail):

>>> import nuad.vienna_nupack as nv
>>> nv.pfunc('GCGCGCGCGC')  # test NUPACK 4
-1.9079766874655928
>>> nv.rna_duplex_multiple([('GCGCGCGCGC', 'GCGCGCGCGC')]) # test ViennaRNA
[-15.7]

Data model

nuad allows one to go from a design with abstract "domains", such as a, a*, b, b*, to concrete DNA sequences, for example, a = 5'-CCCAA-3', a* = 3'-GGGTT-5', b = 5'-AAAAAAC-3', b* = 3'-TTTTTTG-5', obeying the constraints you specify.

There are some pre-built constraints, for example limiting the number of G's in a domain or checking the complex free energy of a strand (i.e., the strand's quantitative amount of "secondary structure") according to the 3rd-party tools NUPACK and ViennaRNA. The user can also specify custom constraints.

In more detail, there are five main types of objects you create to describe your system:

• Domain: A Domain represents a contiguous subsequence of DNA. A single Domain represents both the DNA sequence and its complement. For instance there is one Domain with name a, with two versions: unstarred (a) and starred (a*). If the DNA sequence of a is 5'-CCCAA-3', then the DNA sequence of a* is 3'-GGGTT-5'.

• DomainPool: Each Domain is assigned a single DomainPool, which can be thought of as a "source of DNA sequences" for the Domain. (See exceptions below.) The sequence design algorithm will take DNA sequences from this source when attempting to find DNA sequences to assign to Domain's to satisfy all constraints. Each DomainPool has a fixed length. Since each Domain only has one DomainPool, this means that each Domain has a fixed length as well. If no other constraints are specified, then the DomainPool simply provides all DNA sequences of that length. Though you will generally not call this method yourself, the method DomainPool.generate_sequence() returns a sequence from the pool. This method can be called infinitely many times (i.e., sequences can repeat, though the exact period after which they repeat is an unspecified implementation detail.)

  • There are two types of Domain's with no associated DomainPool. One type is a Domain with the field fixed set to True by calling the method Domain.set_fixed_sequence(), which has some fixed DNA sequence that cannot be changed. A fixed Domain has no DomainPool.)

  • The other type is a Domain with the field dependent set the True (by assigning the field directly). Such a domain is dependent for its sequence on the sequence of some other Domain with dependent = False that either contains it as a subsequence, or is contained in it as a subsequence. For example, one can declare the domain a is independent (has dependent = False), with length 8, and has dependent subdomains b and c of length 5 and 3. a would have a DomainPool, and if a is assigned sequence AAACCGTT, then b is automatically assigned sequence AAACC, and c is automatically assigned sequence GTT. Such subdomains are assigned via the field Domain.subdomains; see the API documentation for more details: https://nuad.readthedocs.io/en/latest/#constraints.Domain.dependent and https://nuad.readthedocs.io/en/latest/#constraints.Domain.subdomains.

• Strand: A Strand contains an ordered list domains of Domain's, together with an identification of which Domain's are starred in this Strand, the latter specified as a set starred_domain_indices of indices (starting at 0) into the list domains. For example, the Strand consisting of Domain's a, b*, c, b, d*, in that order, would have domains = [a, b, c, b, d] and starred_domain_indices = {1, 4}.

• Design: This describes the whole system. Generally you will have one Design, which is composed of a list of Strand's.

• Constraint: There are several kinds of constraint objects. Not all of them are related in the type hierarchy.

  • "hard" constraints on Domain sequences: These are the strictest constraints, which do not even allow certain Domain sequences to be considered, known as "filters". They are applied by a DomainPool before allowing a sequence to be returned from DomainPool.generate_sequence(), which is the method called whenever the search algorithm wants to try a new DNA sequence for a Domain. These are of two types of filters: NumpyFilter and SequenceFilter. Each of them indicates whether a DNA sequence is allowed or not; for instance a filter forbidding 4 G's in a row would permit AGGGTT but forbid AGGGGT. The difference between them is that a NumpyFilter operates on many DNA sequences at a time, representing them as a 2D numpy byte array (e.g., a 1000 × 15 array of bytes to represent 1000 sequences, each of length 15), and for operations that numpy is suited for, can evaluate these filters much faster than the equivalent Python code that would loop over each sequence individually. However, if you have a filter that is not straightforward to express using numpy operations, then a SequenceFilter can be used to express it in plain Python. A SequenceFilter is simply a type alias for a Python function that takes a string as input representing the DNA sequence and returns a Boolean indicating whether the sequence satisfies the filter. Due to the speed of numpy, it is advised to use SequenceFilter's only if necessary because it cannot be expressed as a NumpyFilter.

  • "soft" constraints: All other constraints are subclasses of the abstract superclass Constraint. These constrains are "softer" than filters as described above: sequences violating the constraints are allowed to be assigned to Domain's. The sequence design algorithm steadily improves the design by changing sequences until all of these constraints are satisfied. The different subtypes of the base class Constraint correspond to different parts of the Design that are being evaluated by the Constraint. The types are:

    • SingularConstraint: This is an abstract superclass of the following concrete subclasses. The difference with the other abstract superclass BulkConstraint is explained in BulkConstraint below.

      • DomainConstraint: This only looks at a single Domain. In practice this is not used much, since there's not much information in a Domain other than its DNA sequence, so a SequenceConstraint or NumpyConstraint typically would already have filtered out any DNA sequence not satisfying such a constraint.

      • StrandConstraint: This evaluates a whole Strand. A common example is that NUPACK's pfunc should indicate a complex free energy above a certain threshold, indicating the Strand has little secondary structure. This example constraint is available in the library by calling nupack_strand_complex_free_energy_constraint.

      • DomainPairConstraint: This evaluates a pair of Domain's.

      • StrandPairConstraint: This evaluates a pair of Strand's.

      • ComplexConstraint: This evaluates a tuple of Strand's of arbitrary size.

    • BulkConstraint: The subclasses of SingularConstraint discussed above each evaluate a single part of the design at a time. The classes DomainsConstraint, StrandsConstraint, DomainPairsConstraint, StrandPairsConstraint, ComplexesConstraint are subclasses of BulkConstraint. The difference is that some checks may be faster to do in batch or parallel than one at a time. For instance, RNAduplex, an executable included with ViennaRNA, can examine many pairs of sequences, and it is much faster to give it all pairs at once in a single call to RNAduplex, than to repeatedly call RNAduplex from a Python loop, once for each pair.

    • DesignConstraint: This is rarely used in practice, but it can be used to express any constraint not captured by one of the constraints already listed. It takes the entire design as input.

  The SingularConstraint subclasses DomainsConstraint, StrandsConstraint, DomainPairsConstraint, StrandPairsConstraint, and ComplexConstraint each are given a function evaluate, which takes as input the relevant part of the design (e.g., a StrandPairConstraint takes as input two Strand's; technically the input is a bit more complex; see Parallelism below for details.). evaluate returns a pair (excess, summary), where excess is floating-point value and summary is a string.

  The interpretation of excess is as follows: if the constraint is satisfied, excess should be 0.0. If the constraint is violated, excess should be a positive number indicating "how much" the constraint is violated. The pre-packaged constraints are mostly of the form "compare some numeric value returned by NUPACK or ViennaRNA to a fixed threshold, and return the difference". For example, with a threshold of -1.6 kcal/mol (i.e., we want all strands to have complex free energy greater than or equal to -1.6 kcal/mol), if a Strand has complex free energy of -2.9 kcal/mol according to NUPACK's pfunc, then the excess will be 1.3 = -1.6 - (-2.9), since the actual energy is 1.3 beyond the threshold. If the actual energy is -1.2 instead of -2.9, then it will return 0.0, since it is on the "good" side of the threshold. (The nuad sequence design algorithm actually converts all negative values to 0.0, so one could simply return the value threshold - energy.)

  The second returned value summary is a 1-line string briefly summarizing the result of evaluating the constraint. The pre-packaged constraint that evaluate's a Strand's complex free energy with NUPACK, in the example above, returns summary = "-2.9 kcal/mol". The summary is used in automatically generating reports on the constraints during the search, so that the user can inspect how well the search is doing.

  The BulkConstraint subclasses DomainsConstraint, StrandsConstraint, DomainPairsConstraint, StrandPairsConstraint use a different function, called evaluate_bulk, which take as input a list of "Design parts" (e.g., a list of pairs of Strand's for a StrandPairsConstraint). The return value is of type List[Tuple[DesignPart, float, str]], i.e., a list of triples, where each triple is (part, excess, summary).

  part is the individual part of the design that caused a problem. Generally it will be one of the elements of the list passed to evaluate_bulk, though the returned list could be smaller than the input list. This is because some parts may satisfy the constraint, and generally the only parts returned from evaluate_bulk are those that violated the constraint. excess and summary have the same interpretation as with the "singular" constraints.

  The search algorithm evaluates the constraints, and for each violated constraint, it turns the excess value into a "score" by first passing it through the "score transfer function", which by default squares the value, and then multiplies by the value Constraint.weight (by default 1). The goal of the search is to minimize the sum of scores across all violated Constraint's. The reason that the score is squared is that this leads the search algorithm to (slightly) favor reducing the excess of constraint violations that are "more in excess", i.e., it would reduce the total score more to reduce an excess from 4 to 3 (reducing the score from 4²=16 to 3²=9, a reduction of 16-9=7) than to reduce an excess from 2 to 1 (which reduces 2²=4 to 1²=1, a reduction of only 4-1=3).

  The full search algorithm is described in the API documentation for the function nuad.search.search_for_sequences.

Constraint evaluations must be pure functions of their inputs

For all constraints, it is critical that the evaluate or evaluate_bulk functions be pure functions of their inputs: the return value should depend only on the parameters passed to the function. For example, a StrandPairConstraint takes two strands as input, and its Result return values should depend only on those two strands. Similarly, a StrandsConstraint, whose evaluate_bulk function takes a list of strands as input, should return a list of tuples, where each tuple represents a violation of a strand that depends only on that strand. This is required because nuad does an optimization in which constraints are only evaluated if they depend on parts of the design that contain the domain(s) that changed in the current iteration.

For example, suppose there are 100 strands, but only 3 strands contain the domain x, and x is the domain whose DNA sequence is changed in the current search iteration. Then each StrandConstraint s will be evaluated only on those 3 strands, on the assumption that the other 97 strands would have the same output of the function s.evaluate as before.

In the case of evaluate_bulk, the constraint is even stronger. evaluate_bulk takes a list of objects as input and returns of list of the same size. The i'th element of the returned list should depend only on the i'th element of the input list. This is because a similar optimization is done as above. For example, if the changed domain appears in only 3 strands out of 100, a StrandsConstraint will pass in only those 3 strands as input to evaluate_bulk, not the full list of 100 strands, on the assumption that the other 97 strands would be processed the same by the evaluate_bulk function.

Examples

Some example scripts can be found in the examples/ subfolder.

In particular, the example sst_canvas.py shows a fairly simple design with realistic constraints for designing DNA sequences for a 2D canvas of single-stranded tiles (SSTs), similar to the sort of design from this paper.

Parallelism

Each "singular" constraint in nuad includes the ability to specify the Boolean field Constraint.parallel. If True, then the various parts of the Design will be evaluated in parallel, taking advantage of multi-core systems. In practice, we have found that the overhead associated with doing this is fairly hefty, so it is unlikely that one would see, for example, an 8-fold speedup on an 8-core system. However, one could potentially see a speedup of 2x or 3x.

Given the nature of the stochastic local search, we have found that it is often a more effective and low-overhead use of multiple cores to simply start many independent instances of the designer, each with Constraint.parallel = False for all of the Constraint's. One reason this approach is preferable is that, with very tight constraints that are difficult to satisfy, many runs of the designer will get stuck in local minima, and one often will simply pick the run that got stuck in the lowest minimum.

However, the parallelism feature is there if desired, and it is the reason that the evaluate function described in the Data model section above take slightly more complicated arguments than hinted in that section, which we explain next.

Each "singular" constraint DomainsConstraint, StrandsConstraint, DomainPairsConstraint, StrandPairsConstraint, or ComplexConstraint is specified primarily by a function called evaluate that takes two major types of arguments: DNA sequence(s) of the related design part(s), and optional arguments containing the part(s) themselves. For example, a StrandConstraint's evaluate function takes as input a tuple of strings of length 1, containing the DNA sequence corresponding to the Strand object, as well as the Strand object themselves. For concreteness, we stick with this example of StrandConstraint, but the idea applies to the other singular constraints above.

The type of the Strand parameter is actually Optional[Strand], so could have the value None, for the following reason. If you use nuad's automatic parallelization feature (by setting a Constraint's parallel field to True), then when constraints such as this are called, the Strand's will not be given to the evaluate function, only the DNA sequences. This is because the pathos library is used for parallelization, which uses the dill library to serialize objects for parallel processing. In practice it takes much longer to serialize the entire Strand object than only its DNA sequence. The upshot is that if you don't use parallelization, then you can write constraints that reference not only the DNA sequence of a Strand or other part of the design, but the object representing the part itself. However, to use parallelization, only the DNA sequence of the part will be available to evaluate the constraint, and the second argument representing the part itself will be None.

The evaluate_bulk function for "plural" constraints DomainsConstraint, StrandsConstraint, DomainPairsConstraint, and StrandPairsConstraint does not have this issue, since there is no automatic parallelization feature for "plural" constraints. Therefore it simply takes as input a list of the design "parts" (e.g., list of Strand's, list of pairs of Domain's, etc.) to be evaluated.

Reporting issues

Please report issues (bugs or feature requests) at the nuad GitHub repository issues page.

Contributing

See the CONTRIBUTING document.

NUPACK Copyright Notice

Since nuad will eventually be distributed with NUPACK, we include the following license agreement as required by NUPACK.

NUPACK Software License Agreement for Non-Commercial Academic Use and Redistribution

Copyright © 2022 California Institute of Technology. All rights reserved.

1. Use and redistribution in source form and/or binary form, with or without modification, are permitted for non-commercial academic purposes only, provided that the following conditions are met:

2. Redistributions in source form must retain the above copyright notice, this list of conditions and the following disclaimer.

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4. Web applications that use the software in source form or binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in online documentation provided with the web application.

Neither the name of the copyright holder nor the names of its contributors may be used to endorse or promote derivative works without specific prior written permission.

Disclaimer

This software is provided by the copyright holders and contributors "as is" and any express or implied warranties, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose are disclaimed. In no event shall the copyright holder or contributors be liable for any direct, indirect, incidental, special, exemplary, or consequential damages (including, but not limited to, procurement of substitute goods or services; loss of use, data, or profits; or business interruption) however caused and on any theory of liability, whether in contract, strict liability, or tort (including negligence or otherwise) arising in any way out of the use of this software, even if advised of the possibility of such damage.