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Team:AMU-Poznan/Project
From 2014.igem.org
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Sh-miR designer is a project we started during iGEM 2013. This year we would like to continue and expand functionality of the software. sh-miR designer v1.0 (link) is aimed to create sh-miR molecules based on siRNAs provided by the user. In sh-miR designer v2.0 only the mRNA number (from NCBI database), which expression should be decreased can be provided. Moreover, we expanded functionality of the software with off-target validation and check of immune motifs and also extended miRNA-shuttles database. | Sh-miR designer is a project we started during iGEM 2013. This year we would like to continue and expand functionality of the software. sh-miR designer v1.0 (link) is aimed to create sh-miR molecules based on siRNAs provided by the user. In sh-miR designer v2.0 only the mRNA number (from NCBI database), which expression should be decreased can be provided. Moreover, we expanded functionality of the software with off-target validation and check of immune motifs and also extended miRNA-shuttles database. | ||
sh-miR Designer is a software aimed for fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research. | sh-miR Designer is a software aimed for fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research. | ||
| - | We will provide two applications to access the software, one which require siRNA sequences and the second which require transcript accession number from NCBI database. Each user will receive an account with login (e-mail) and password where he/she would be able to check history of software usage. | + | We will provide two applications to access the software, one which require siRNA sequences and the second which require transcript accession number from NCBI database. Each user will receive an account with login (e-mail) and password where he/she would be able to check history of software usage.</br> |
| - | < | + | <h1>Biological background</h1> |
| - | < | + | </br> |
| - | + | RNA interference (RNAi) is a physiological process of posttranscriptional gene expression regulation caused by double stranded RNA molecules. This is one of the most willingly used experimental techniques. It is used in experiments designed to explore gene function, in creation cellular models of diseases or in genetic therapy design. Among RNAi technology reagents are siRNA (short interfering RNA) and vector reagents shRNA (short hairpin RNA) and sh-miR (sh- microRNA). RNAi reagents are integrated into endogenous RNAi pathway on different phases. sh-miR reagents are designed in the way to be similar to endogenous miRNA precursors (pri-miRNA), because it is important feature to be recognized by proteins involved in microRNA (miRNA) biogenesis.</br> | |
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/e/e1/Dicer.png"> |
| + | </br> | ||
| + | shRNA or sh-miR reagents are introduced into cells with usage of genetic vectors – plasmids or viral vectors to ensure intracellular expression. They have huge therapeutic potential beacuse of stable reagent production in cells what enables one time therapeutic dosage. Usage of synthetic siRNA reagents requires often dosage, what makes therapy more difficult, especially in case of Central Nervous System Diseases.</br> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/a/a8/Jaja.png"> | ||
| + | </br> | ||
| + | |||
| + | It was recently shown that RNAi reagents introduces into cells can trigger unwanted non-specific effects, e.g. can interact with different transcripts besides the desired one (so called off-target effect) or induce immunological effect. Because in processing of sh-miR reagents endogenous endonucleasis Drosha and Dicer are involved, in case of high expression of reagent they can saturate those proteins as well as Exportin-5 transport protein responsible for export of sh-miR from nucleus. The saturation can influence endogenous RNAi mechanism. It was shown that sh-miR reagents influence less endogenous RNAi pathway, because of lower level of produced double-stranded RNA (dsRNA), preserving efficiency of translation silencing. They also show longer effect than different types of RNAi reagents. Because of described features sh-miR reagents seems to be good choice when designing efficient and non-toxic RNAi reagents.</br></br> | ||
| + | |||
| + | sh-miR Designer software was designed sh-miR Designer is a software aimed for fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Until now scientists have to use multiple unconnected software to design single sh-miR molecule. It is time consuming process, what limits number of designed molecules for biological testing. There is no such software, which includes all important features as molecule spatial structure. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. This interdisciplinary project includes computer sciences with biomedical science. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research. </br></br> | ||
| + | |||
| + | </br> | ||
| + | </br> | ||
<h1>Standard Parts</h1> | <h1>Standard Parts</h1> | ||
</br> | </br> | ||
| - | We are creating a particle which can be inserted into plasmid built by Standard parts from Registry of Standard Biological Parts. | + | We are creating a particle which can be inserted into plasmid built by Standard parts from Registry of Standard Biological Parts. The sh-miR molecules are inserted into plasmid backbone downstream chosen promoter. You can add any regulatory sequence you want or maybe a fragment coding fluorescent protein to see if the transfection was succesfull. The molecules are fully compatibile with Standard Biological Parts - what is important for users from iGEM especially :). |
<img src="https://static.igem.org/mediawiki/2014/b/bb/Shmir-STANDARD.png" width="60%"></br> | <img src="https://static.igem.org/mediawiki/2014/b/bb/Shmir-STANDARD.png" width="60%"></br> | ||
| + | <img src="http://biosettia.com/images/miRNA/lentiviral-mirna-expression-01.png" width="60%"></br> | ||
| + | picture from http://biosettia.com/ | ||
<h1>User Guide</h1> | <h1>User Guide</h1> | ||
<div align="left"> | <div align="left"> | ||
| + | There are two suplementary versions of our software. First one (based on last year's software sh-miR v1.0) enable to design sh-miR molecules based on siRNA sequence and the second design sh-miR molecules when NCBI transcript number (NM_...) is provided. In the second version the user also can set GC content, maximum off-target and immunostimulatory properties. The user can also set which pri-miRNA should be used to design sh-miR (or all available in our miRNA database).</br> | ||
| + | |||
| + | Please follow video where you can see how to use web version of the software</br> | ||
| + | and short description how to use client version of the software</br> | ||
| + | Both versions are well documented on github and readthedocs (see below)</br> | ||
<h3>Usage via Web server</h1> | <h3>Usage via Web server</h1> | ||
| - | go to our website <a href="shmir.wmi.amu.edu.pl">shmir.wmi.amu.edu.pl</a> | + | go to our website <a href="https://shmir.wmi.amu.edu.pl">shmir.wmi.amu.edu.pl</a> |
| + | <div align = "center"> | ||
| + | <iframe width="420" height="315" | ||
| + | src="https://www.youtube.com/embed/D3DO_5NODQo"> | ||
| + | </iframe> | ||
| + | </div></br></br> | ||
| + | <br/>input form:<br/></br></br> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/2/24/Zrzut_ekranu_2014-10-15_o_18.07.36.png" width="60%"></br></br></br> | ||
| + | <br/>results:<br/></br></br> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/8/8a/Zrzut_ekranu_2014-10-15_o_22.19.56.png" width="60%"></br></br></br> | ||
| + | users' history:<br/></br></br> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/c/c2/Zrzut_ekranu_2014-10-15_o_18.09.10.png" width="60%"><br/></br></br> | ||
<h3>Usage via client</h3> | <h3>Usage via client</h3> | ||
</br>Fold sequence via mfold:</br> | </br>Fold sequence via mfold:</br> | ||
| Line 96: | Line 125: | ||
</br> | </br> | ||
<h1>API documentation</h1> | <h1>API documentation</h1> | ||
| - | </br>Here you can find | + | </br>Here you can find documentation from code:</br> |
| + | |||
| + | API: <a href="https://shmir-api.rtfd.org">shmir-api.rtfd.org</a></br> | ||
| + | Client: <a href="https://shmir-client.rtfd.org">shmir-client.rtfd.org</a></br> | ||
| + | Webpage: <a href="https://shmir-designer.rtfd.org">shmir-designer.rtfd.org</a></br> | ||
| + | |||
| + | |||
<h1>API for developers</h1> | <h1>API for developers</h1> | ||
</br> | </br> | ||
| Line 102: | Line 137: | ||
<h1>Releases (re-use of 2013iGEM software)</h1> | <h1>Releases (re-use of 2013iGEM software)</h1> | ||
<div align="left"> | <div align="left"> | ||
| - | </br>sh-miR designer v1.0 </br> | + | <h3></br>sh-miR designer v1.0 </br></h3> |
First version of the program presented on iGEM 2013 by our team. The user have to provide siRNA sequence (one or both strands). The miRNA database includes 5 miRNAs. </br> | First version of the program presented on iGEM 2013 by our team. The user have to provide siRNA sequence (one or both strands). The miRNA database includes 5 miRNAs. </br> | ||
| - | sh-miR designer v1.1 </br> | + | <h3>sh-miR designer v1.1 </br></h3> |
Strand discrimination functionality was added based on miRbase. </br> | Strand discrimination functionality was added based on miRbase. </br> | ||
extended miRNA database </br> | extended miRNA database </br> | ||
| - | sh-miR designer v2.0 </br> | + | <h3>sh-miR designer v2.0 </br></h3> |
siRNA prediction algorithm was added to functionality (the input is NCBI transcript number) </br> | siRNA prediction algorithm was added to functionality (the input is NCBI transcript number) </br> | ||
user can choose GC content of siRNA molecule, maximal off-target, if he/she wants immunostimulatory sequences inside the molecule and to choose if he/she wants to include all or chosen scaffold </br> | user can choose GC content of siRNA molecule, maximal off-target, if he/she wants immunostimulatory sequences inside the molecule and to choose if he/she wants to include all or chosen scaffold </br> | ||
| Line 120: | Line 155: | ||
</br> | </br> | ||
<div align="left"> | <div align="left"> | ||
| + | |||
In our application we are using unit tests to test alghoritms, functions and database.</br> | In our application we are using unit tests to test alghoritms, functions and database.</br> | ||
We are testing inputs and outputs:</br> | We are testing inputs and outputs:</br> | ||
| + | <pre> | ||
def test_validate_gc_content(self):</br> | def test_validate_gc_content(self):</br> | ||
is_in_range = validate_gc_content('ACGT', 40, 60)</br> | is_in_range = validate_gc_content('ACGT', 40, 60)</br> | ||
| Line 138: | Line 175: | ||
self.assertEqual(len(objs), 1)</br> | self.assertEqual(len(objs), 1)</br> | ||
</br> | </br> | ||
| + | </pre> | ||
We are using Mock library to test our functions which are using external API’s to create fake response from server and return them into our function:</br> | We are using Mock library to test our functions which are using external API’s to create fake response from server and return them into our function:</br> | ||
| + | <pre> | ||
@patch.object(ncbi_api.Entrez, 'esearch')</br> | @patch.object(ncbi_api.Entrez, 'esearch')</br> | ||
@patch.object(ncbi_api.Entrez, 'efetch')</br> | @patch.object(ncbi_api.Entrez, 'efetch')</br> | ||
| Line 154: | Line 193: | ||
)</br> | )</br> | ||
</br> | </br> | ||
| + | </pre> | ||
</div> | </div> | ||
<h1>Validation</h1> | <h1>Validation</h1> | ||
| Line 164: | Line 204: | ||
user 0m0.155s</br> | user 0m0.155s</br> | ||
sys 0m0.041s</br> | sys 0m0.041s</br> | ||
| + | </br> | ||
| + | </br> | ||
| + | <h3>Designed sh-miR</h3> | ||
| + | </br></br> | ||
| + | for $ ./shmir_client.py from_transcript NM_000546.5 | ||
| + | </br> | ||
| + | 1: backbone: miR-31, score: 106, sequence: ACATGACGGAGGTTGTGAG</br> | ||
| + | pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/7270b2d2-0aca-472b-853e-5a9631164d46</br> | ||
| + | result: </br>CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGACATGACGGAGGTTGTGAGCGTTGAACTGGGAACCCTCACAACCTCCGTCA | ||
| + | </br>TGTCTCTCCAGTTCCGAGTTACAGGAGCAATACCAT</br>CCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT | ||
| + | </br></br> | ||
| + | 2: backbone: miR-31, score: 106, sequence: ATGTTTTGTAGAGATGGGG</br> | ||
| + | pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/56232916-2649-4eb4-9970-c0b0a08ce64e</br> | ||
| + | result: </br>CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATGTTTTGTAGAGATGGGGCGTTGAACTGGGAACCCCCCATCTCTACA | ||
| + | </br>AAACATCTCTCCAGTTCCGAGTTACAGGAGCAATACCAT</br>CCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT | ||
| + | </br></br> | ||
| + | 3: backbone: miR-31, score: 106, sequence: ATTACATGGGGTCTAGAAC</br> | ||
| + | pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/37cd84aa-362c-484d-8681-929043cc9c61</br> | ||
| + | result: </br>CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATTACATGGGGTCTAGAACCGTTGAACTGGGAACCGTTCTAG | ||
| + | </br>ACCCCATGTAATCTCTCCAGTTCCGAGTTACAGGAGCAATACCAT</br>CCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT | ||
| + | </br></br> | ||
| + | 4: backbone: miR-31, score: 106, sequence: ACCATCATCACACTGGAAG</br> | ||
| + | pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/50b763f6-2193-4743-96c0-61601880e574</br> | ||
| + | result: </br>CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGACCATCATCACACTGGAAGCGTTGAACTGGGAACCCTT | ||
| + | </br>CCAGTGTGATGATGGTCTCTCCAGTTCCGAGTTACAGGAGCAATACCAT</br>CCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT | ||
| + | </br></br> | ||
| + | 5: backbone: miR-31, score: 106, sequence: ATCTCACCCCATCCCACAC</br> | ||
| + | pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/019ce915-0282-4d83-9538-93c36c790fa8</br> | ||
| + | result: </br>CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATCTCACCCCATCCCACACCGTTGAAC | ||
| + | </br>TGGGAACCGTGTGGGATGGGGTGAGATCTCTCCAGTTCCGAGTTACAGGAGCAATACCAT</br>CCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT</br> | ||
| + | </br> | ||
| + | see example pdf with structure <a href="https://static.igem.org/mediawiki/2014/d/da/Sequence_1.pdf">link</a> | ||
| + | </br> | ||
| + | During sh-miR designer development, colleagues from befriended lab used our software, so we had constant feedback from potential users,</br> | ||
| + | who by the way, have nothing to do with bioinformatics. So it's really hard to satisfy their strong need for easy interface.</br> | ||
| + | We also had significant feedback from our survey (see Modeling tab), so we knew what we should emphasise on during develoment.</br> | ||
| + | |||
| + | </br> | ||
<h1>Test cases</h1> | <h1>Test cases</h1> | ||
</br>1) ATXN3 - therapeutic sh-miR against SCA3 disease</br> | </br>1) ATXN3 - therapeutic sh-miR against SCA3 disease</br> | ||
| Line 252: | Line 330: | ||
<div align="left">Bronze. The following 4 goals must be achieved:</br> | <div align="left">Bronze. The following 4 goals must be achieved:</br> | ||
<ul> | <ul> | ||
| - | <li><b>Register the team, have a great summer, and have fun attending the Jamboree in Boston.</br>< | + | <li><b>Register the team, have a great summer, and have fun attending the Jamboree in Boston.</br></li> |
| - | <li>Create and share a description of the team's project via the iGEM wiki.</br></b>< | + | <li>Create and share a description of the team's project via the iGEM wiki.</br></b></li> |
| - | <li>Present a Poster and Talk at the Regional Jamboree and World Championship Jamboree.<b> We will on Jamboree :)</br>< | + | <li>Present a Poster and Talk at the Regional Jamboree and World Championship Jamboree.<b> We will on Jamboree :)</br></li> |
| - | <li>Develop and make available, via The iGEM Software Registry, an open source software that supports Synthetic Biology based on Standard Parts.< | + | <li>Develop and make available, via The iGEM Software Registry, an open source software that supports Synthetic Biology based on Standard Parts.</li></b></br></br> |
</ul> | </ul> | ||
Silver: In addition to the Bronze Medal requirements, the following 4 goals must be achieved:</br> | Silver: In addition to the Bronze Medal requirements, the following 4 goals must be achieved:</br> | ||
| Line 265: | Line 343: | ||
</ul> | </ul> | ||
Gold: In addition to the Bronze and Silver Medal requirements, two additional goals must be achieved: </br> | Gold: In addition to the Bronze and Silver Medal requirements, two additional goals must be achieved: </br> | ||
| - | <b>Provide a convincing validation, testing the performance of the development -- experimentally (can be outsourced) or by other teams and users. Note, even if the algorithm or tool turns out not to work that well, the Gold requirement is fulfilled if the test is good and the analysis convincing. Validation may include: preferably experiments, informatics analysis (complexity, run time) of an algorithm, or user studies.</br></b> | + | <b><ul><li>Provide a convincing validation, testing the performance of the development -- experimentally (can be outsourced) or by other teams and users. Note, even if the algorithm or tool turns out not to work that well, the Gold requirement is fulfilled if the test is good and the analysis convincing. Validation may include: preferably experiments, informatics analysis (complexity, run time) of an algorithm, or user studies.</li></ul></br></b> |
And the second goal can be any one of the following:</br> | And the second goal can be any one of the following:</br> | ||
<ul> | <ul> | ||
Latest revision as of 21:18, 17 October 2014
sh-miR Designer v2.0Sh-miR designer is a project we started during iGEM 2013. This year we would like to continue and expand functionality of the software. sh-miR designer v1.0 (link) is aimed to create sh-miR molecules based on siRNAs provided by the user. In sh-miR designer v2.0 only the mRNA number (from NCBI database), which expression should be decreased can be provided. Moreover, we expanded functionality of the software with off-target validation and check of immune motifs and also extended miRNA-shuttles database. sh-miR Designer is a software aimed for fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research. We will provide two applications to access the software, one which require siRNA sequences and the second which require transcript accession number from NCBI database. Each user will receive an account with login (e-mail) and password where he/she would be able to check history of software usage.Biological backgroundRNA interference (RNAi) is a physiological process of posttranscriptional gene expression regulation caused by double stranded RNA molecules. This is one of the most willingly used experimental techniques. It is used in experiments designed to explore gene function, in creation cellular models of diseases or in genetic therapy design. Among RNAi technology reagents are siRNA (short interfering RNA) and vector reagents shRNA (short hairpin RNA) and sh-miR (sh- microRNA). RNAi reagents are integrated into endogenous RNAi pathway on different phases. sh-miR reagents are designed in the way to be similar to endogenous miRNA precursors (pri-miRNA), because it is important feature to be recognized by proteins involved in microRNA (miRNA) biogenesis.
shRNA or sh-miR reagents are introduced into cells with usage of genetic vectors – plasmids or viral vectors to ensure intracellular expression. They have huge therapeutic potential beacuse of stable reagent production in cells what enables one time therapeutic dosage. Usage of synthetic siRNA reagents requires often dosage, what makes therapy more difficult, especially in case of Central Nervous System Diseases.
It was recently shown that RNAi reagents introduces into cells can trigger unwanted non-specific effects, e.g. can interact with different transcripts besides the desired one (so called off-target effect) or induce immunological effect. Because in processing of sh-miR reagents endogenous endonucleasis Drosha and Dicer are involved, in case of high expression of reagent they can saturate those proteins as well as Exportin-5 transport protein responsible for export of sh-miR from nucleus. The saturation can influence endogenous RNAi mechanism. It was shown that sh-miR reagents influence less endogenous RNAi pathway, because of lower level of produced double-stranded RNA (dsRNA), preserving efficiency of translation silencing. They also show longer effect than different types of RNAi reagents. Because of described features sh-miR reagents seems to be good choice when designing efficient and non-toxic RNAi reagents.
sh-miR Designer software was designed sh-miR Designer is a software aimed for fast and efficient design of effective RNA interference (RNAi) reagents - sh-miRs, also known as artificial miRNAs. sh-miRs are RNA particles whose structure is based on miRNA precursor pri-miRNA, but sequence interacting with transcript is changed depending on research purpose. Until now scientists have to use multiple unconnected software to design single sh-miR molecule. It is time consuming process, what limits number of designed molecules for biological testing. There is no such software, which includes all important features as molecule spatial structure. Maintenance of structure of pri-miRNA is very important to enable cellular processing and therefore ensure functionality of artificial particles. This interdisciplinary project includes computer sciences with biomedical science. sh-miRs delivered to cells on genetic vectors - plasmids or viral vectors - enter natural RNAi pathway and silence target mRNA. They can be used in genetic therapies and basic biomedical research.
Standard PartsWe are creating a particle which can be inserted into plasmid built by Standard parts from Registry of Standard Biological Parts. The sh-miR molecules are inserted into plasmid backbone downstream chosen promoter. You can add any regulatory sequence you want or maybe a fragment coding fluorescent protein to see if the transfection was succesfull. The molecules are fully compatibile with Standard Biological Parts - what is important for users from iGEM especially :).
picture from http://biosettia.com/
User Guide
There are two suplementary versions of our software. First one (based on last year's software sh-miR v1.0) enable to design sh-miR molecules based on siRNA sequence and the second design sh-miR molecules when NCBI transcript number (NM_...) is provided. In the second version the user also can set GC content, maximum off-target and immunostimulatory properties. The user can also set which pri-miRNA should be used to design sh-miR (or all available in our miRNA database).
Please follow video where you can see how to use web version of the software
and short description how to use client version of the software
Both versions are well documented on github and readthedocs (see below)
Usage via Web server
go to our website shmir.wmi.amu.edu.pl
Technology
The application was built with Python 2.7. It is divided into 3 parts:
RESTful API - the “heart” of our software - it performs all calculations. Our application is built on CentOS (Linux) operating system. To maintain better compatibility with our server we use Vagrant to create Virtual Machine and Puppet to manage infrastructure of our project. All you need to do is: type into console “vagrant up” and after a while everything is ready to work. This subproject is written with following technologies/frameworks/libraries: Flask, Flask-Cache, SqlAlchemy, PostgreSQL, Celery, Flower, Rabbitmq, Redis, BioPython, mfold (lightweight), uwsgi, sphinx, Python testing tools (mock, nose, unittest).
Console client - simple console application which communicates with API using requests library. It has 3 options: mfold (folding structure), from_sirna (create sh-miR from siRNA) and from_transcript (create sh-miR from transcript name).
Website - application written in Django framework. It enables you to use the API via graphical interface. You can also create your own account and save the history of your requests for later usage.
API documentationHere you can find documentation from code: API: shmir-api.rtfd.org Client: shmir-client.rtfd.org Webpage: shmir-designer.rtfd.orgAPI for developersHere you can find api for developers: https://github.com/Nozdi/shmirReleases (re-use of 2013iGEM software)sh-miR designer v1.0First version of the program presented on iGEM 2013 by our team. The user have to provide siRNA sequence (one or both strands). The miRNA database includes 5 miRNAs.sh-miR designer v1.1Strand discrimination functionality was added based on miRbase. extended miRNA databasesh-miR designer v2.0siRNA prediction algorithm was added to functionality (the input is NCBI transcript number) user can choose GC content of siRNA molecule, maximal off-target, if he/she wants immunostimulatory sequences inside the molecule and to choose if he/she wants to include all or chosen scaffold extended miRNA database What we are working on: strand discrimination based on therodynamics of siRNA ends; preparing construct with flanking restriction enzymes providing plasmids with parts from standard partsUnit Tests
In our application we are using unit tests to test alghoritms, functions and database.
We are testing inputs and outputs:
def test_validate_gc_content(self):
is_in_range = validate_gc_content('ACGT', 40, 60)
self.assertTrue(is_in_range)
Caught errors:
def multiple_input(self, data, error):
for strand in data:
with self.assertRaises(errors.InputException) as err:
validators.check_input(strand)
self.assertEqual(error, str(err.exception))
Database models:
def test_get_and_put_to_db(self):
new_obj = self.TestModel(id=153)
self.put_to_db(new_obj)
objs = self.get_all(self.TestModel)
self.assertEqual(len(objs), 1)
We are using Mock library to test our functions which are using external API’s to create fake response from server and return them into our function:
@patch.object(ncbi_api.Entrez, 'esearch')
@patch.object(ncbi_api.Entrez, 'efetch')
def test_get_data(self, mock_efetch, mock_esearch):
ids = [69]
mock_read = Mock()
mock_read.read.return_value = json.dumps({'esearchresult': {'idlist': ids}})
mock_esearch.return_value = mock_read
ncbi_api.get_data(self.GOOD_MRNA)
mock_esearch.assert_called_with(
term='NM_001128164.1', db=self.DATA_BASE, retmode='json'
)
mock_efetch.assert_called_once_with(
db=self.DATA_BASE, id=ids, rettype='fasta', retmode='text'
)
Validationold version shmiR v1.0 (average time)real 0m6.854s user 0m0.063s sys 0m0.027snew version shmiR v1.1 (average times)real 0m3.271s user 0m0.155s sys 0m0.041sDesigned sh-miRfor $ ./shmir_client.py from_transcript NM_000546.5 1: backbone: miR-31, score: 106, sequence: ACATGACGGAGGTTGTGAG pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/7270b2d2-0aca-472b-853e-5a9631164d46 result: CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGACATGACGGAGGTTGTGAGCGTTGAACTGGGAACCCTCACAACCTCCGTCA TGTCTCTCCAGTTCCGAGTTACAGGAGCAATACCATCCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT 2: backbone: miR-31, score: 106, sequence: ATGTTTTGTAGAGATGGGG pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/56232916-2649-4eb4-9970-c0b0a08ce64e result: CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATGTTTTGTAGAGATGGGGCGTTGAACTGGGAACCCCCCATCTCTACA AAACATCTCTCCAGTTCCGAGTTACAGGAGCAATACCATCCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT 3: backbone: miR-31, score: 106, sequence: ATTACATGGGGTCTAGAAC pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/37cd84aa-362c-484d-8681-929043cc9c61 result: CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATTACATGGGGTCTAGAACCGTTGAACTGGGAACCGTTCTAG ACCCCATGTAATCTCTCCAGTTCCGAGTTACAGGAGCAATACCATCCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT 4: backbone: miR-31, score: 106, sequence: ACCATCATCACACTGGAAG pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/50b763f6-2193-4743-96c0-61601880e574 result: CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGACCATCATCACACTGGAAGCGTTGAACTGGGAACCCTT CCAGTGTGATGATGGTCTCTCCAGTTCCGAGTTACAGGAGCAATACCATCCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT 5: backbone: miR-31, score: 106, sequence: ATCTCACCCCATCCCACAC pdf: ./results/transcript/e9e8b9a2-7e3b-4b24-b26a-633332a3f6b2/miR-31/019ce915-0282-4d83-9538-93c36c790fa8 result: CATAACAACGAAGAGGGATGGTATTGCTCCTGTAACTCGGAACTGGAGAGGATCTCACCCCATCCCACACCGTTGAAC TGGGAACCGTGTGGGATGGGGTGAGATCTCTCCAGTTCCGAGTTACAGGAGCAATACCATCCCTCTTCGTTGTTATGCTTTCCTGTCTGACAGCAGCTTGGCTACCTCCGTCCTGTTCCTCCTTGTCTT see example pdf with structure link During sh-miR designer development, colleagues from befriended lab used our software, so we had constant feedback from potential users, who by the way, have nothing to do with bioinformatics. So it's really hard to satisfy their strong need for easy interface. We also had significant feedback from our survey (see Modeling tab), so we knew what we should emphasise on during develoment.Test cases1) ATXN3 - therapeutic sh-miR against SCA3 disease transcript number: NM_001164782.1 GC content: default Off-target: 10 scaffold: ALL immunostimulatory: NO 2) P54 - therapeutic sh-miR against cancer transcript number: NM_000546.5 GC content: default Off-target: 10 scaffold: ALL immunostimulatory: YES 3) biological function analysis, PARP1, we have miR-30a in our lab transcript number: NM_001618.3 GC content: default Off-target: 0 scaffold: miR-30a immunostimulatory: no_difference (default) 4) biological function, PARP1, we have miR-30a in our laboratory and have immune cells transcript number: NM_001618.3 GC content: default Off-target: 0 scaffold: miR-30a immunostimulatory: NO 5) biological function, PARP1 and have immune cells transcript number: NM_001618.3 GC content: default Off-target: 0 scaffold: ALL immunostimulatory: NOReferences
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33
Judging Form (in bold what we did to achieve gold medal)Bronze. The following 4 goals must be achieved:
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users' history:
