Name: mirmachine: a one-stop shop for plant mirna annotation
Uploaded: 2021-05-01T13:30:00
Description: Watch this Scientific Journal Video about mirMachine: A One-Stop Shop for Plant miRNA Annotation at JoVE.com

1. Software dependencies and installation
<ol>
	<li>Install software dependencies from their home site or using conda.
	<ol>
		<li>Download and install Perl, if it is not already installed, from its home site (https://www.perl.org/get.html). 
		NOTE: Represented results were predicted using Perl v5.32.0.</li>
		<li>Download Blast+, an alignment program, from its home site (https://www.ncbi.nlm.nih.gov/books/NBK279671/) as an executable and as source code. 
		NOTE: Represented results were predicted using the ...

<ol>
	<li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+biogenesis,+and+activity+of+plant+microRNAs.">Origin, biogenesis, and activity of plant microRNAs.</a> Cell. 136 (4), 669-687 (2009).</li><li>Budak, H., Akpinar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+miRNAs:+biogenesis,+organization+and+origins.">Plant miRNAs: biogenesis, organization and origins.</a> Functional &amp; Integrative Genomics. 15 (5), 523-531 (2015).</li><li>Lee, R. C., Feinbaum, R. L., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+heterochronic+gene+lin-4+encodes+small+RNAs+with+antisense+complementarity+to+lin-14.">The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.</a> Cell. 75 (5), 843-854 (1993).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+plant+MIR168a+specifically+targets+mammalian+LDLRAP1:+evidence+of+cross-kingdom+regulation+by+microRNA.">Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA.</a> Cell Research. 22 (1), 107-126 (2012).</li><li>Pang, K. C., Frith, M. C., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+noncoding+RNAs:+Lack+of+conservation+does+not+mean+lack+of+function.">Rapid evolution of noncoding RNAs: Lack of conservation does not mean lack of function.</a> Trends in Genetics. 22 (1), 1-5 (2006).</li><li>Guleria, P., Mahajan, M., Bhardwaj, J., Yadav, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+small+RNAs:+biogenesis,+mode+of+action+and+their+roles+in+abiotic+stresses.">Plant small RNAs: biogenesis, mode of action and their roles in abiotic stresses.</a> Genomics, Proteomics and Bioinformatics. 9 (6), 183-199 (2011).</li><li>Jones-Rhoades, M. W., Bartel, D. P., Bartel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+their+regulatory+roles+in+plants.">MicroRNAs and their regulatory roles in plants.</a> Annual Review of Plant Biology. 57, 19-53 (2006).</li><li>Singh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+small+RNAs:+advancement+in+the+understanding+of+biogenesis+and+role+in+plant+development.">Plant small RNAs: advancement in the understanding of biogenesis and role in plant development.</a> Planta. 248 (3), 545-558 (2018).</li><li>Lucas, S. J., Budak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+the+wheat+from+the+chaff:+identifying+miRNAs+in+genomic+survey+sequences+of+Triticum+aestivum+chromosome+1AL.">Sorting the wheat from the chaff: identifying miRNAs in genomic survey sequences of Triticum aestivum chromosome 1AL.</a> PloS One. 7 (7), 40859 (2012).</li><li>Li, S., Castillo-Gonz&#225;lez, C., Yu, B., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+plant+small+RNAs+in+development+and+in+stress+responses.">The functions of plant small RNAs in development and in stress responses.</a> Plant Journal. 90 (4), 654-670 (2017).</li><li>Lee, Y., Jeon, K., Lee, J. T., Kim, S., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+maturation:+Stepwise+processing+and+subcellular+localization.">MicroRNA maturation: Stepwise processing and subcellular localization.</a> EMBO Journal. 21 (17), 4663-4670 (2002).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+genes+are+transcribed+by+RNA+polymerase+II.">MicroRNA genes are transcribed by RNA polymerase II.</a> EMBO Journal. 23 (2), 4051-4060 (2004).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+Genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: Genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+RNase+III+Drosha+initiates+microRNA+processing.">The nuclear RNase III Drosha initiates microRNA processing.</a> Nature. 425 (6956), 415-419 (2003).</li><li>Meyers, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+for+annotation+of+plant+microRNAs.">Criteria for annotation of plant microRNAs.</a> Plant Cell. 20 (12), 3186-3190 (2008).</li><li>Sanei, M., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+microRNA+turnover.">Mechanisms of microRNA turnover.</a> Current Opinion in Plant Biology. 27, 199-206 (2015).</li><li>Li, J., Yang, Z., Yu, B., Liu, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+protects+miRNAs+and+siRNAs+from+a+3&#8242;-end+uridylation+activity+in+Arabidopsis.">Methylation protects miRNAs and siRNAs from a 3&#8242;-end uridylation activity in Arabidopsis.</a> Current Biology. 15 (16), 1501-1507 (2005).</li><li>Rogers, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+turnover,+and+mode+of+action+of+plant+microRNAs.">Biogenesis, turnover, and mode of action of plant microRNAs.</a> Plant Cell. 25 (7), 2383-2399 (2013).</li><li>Axtell, M. J., Meyers, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+criteria+for+plant+microRNA+annotation+in+the+Era+of+big+data.">Revisiting criteria for plant microRNA annotation in the Era of big data.</a> Plant Cell. 30 (2), 272-284 (2018).</li><li>Camacho, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAST+:+architecture+and+applications.">BLAST+: architecture and applications.</a> BMC Bioinformatics. 10 (1), 421 (2009).</li><li>Markham, N. R. N., Zuker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNAFold:+Software+for+nucleic+acid+folding+and+hybridization.">UNAFold: Software for nucleic acid folding and hybridization.</a> Methods in Molecular Biology. 453, 3-31 (2008).</li><li>Alptekin, B., Akpinar, B. A., Budak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+prescription+for+plant+miRNA+identification.">A comprehensive prescription for plant miRNA identification.</a> Frontiers in Plant Science. 7, 2058 (2017).</li><li>Zhang, B., Pan, X., Cannon, C. H., Cobb, G. P., Anderson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+divergence+of+plant+microRNA+genes.">Conservation and divergence of plant microRNA genes.</a> Plant Journal. 46 (2), 243-259 (2006).</li><li>Appels, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shifting+the+limits+in+wheat+research+and+breeding+using+a+fully+annotated+reference+genome.">Shifting the limits in wheat research and breeding using a fully annotated reference genome.</a> Science. 361 (6403), 7191 (2018).</li><li>Wang, Y., Kuang, Z., Li, L., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioinformatics+pipeline+to+accurately+and+efficiently+analyze+the+microRNA+transcriptomes+in+plants.">A bioinformatics pipeline to accurately and efficiently analyze the microRNA transcriptomes in plants.</a> Journal of Visualized Experiments: JoVE. (155), e59864 (2020).</li><li>Kozomara, A., Griffiths-Jones, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRBase:+Annotating+high+confidence+microRNAs+using+deep+sequencing+data.">MiRBase: Annotating high confidence microRNAs using deep sequencing data.</a> Nucleic Acids Research. 42, 68-73 (2014).</li><li>Lorenz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ViennaRNA+Package+2.0.">ViennaRNA Package 2.0.</a> Algorithms for Molecular Biology. 6 (1), 26 (2011).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+transposable+elements+on+genome+structure+and+evolution+in+bread+wheat.">Impact of transposable elements on genome structure and evolution in bread wheat.</a> Genome Biology. 19 (1), 103 (2018).</li><li>Flavell, R. B., Bennett, M. D., Smith, J. B., Smith, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+size+and+the+proportion+of+repeated+nucleotide+sequence+DNA+in+plants.">Genome size and the proportion of repeated nucleotide sequence DNA in plants.</a> Biochemical Genetics. 12 (4), 257-269 (1974).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repetitive+landscape+of+the+5100+Mbp+barley+genome.">The repetitive landscape of the 5100 Mbp barley genome.</a> Mobile DNA. 8, 22 (2017).</li><li>Yang, Q., Ye, Q. A., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+siRNA+production+from+repetitive+DNA.">Mechanism of siRNA production from repetitive DNA.</a> Genes and Development. 29 (5), 526-537 (2015).</li><li>Lam, J. K. W., Chow, M. Y. T., Zhang, Y., Leung, S. W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+versus+miRNA+as+therapeutics+for+gene+silencing.">siRNA versus miRNA as therapeutics for gene silencing.</a> Molecular Therapy. Nucleic Acids. 4 (9), 252 (2015).</li><li>Bartel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+directing+siRNA+biogenesis.">MicroRNAs directing siRNA biogenesis.</a> Nature Structural and Molecular Biology. 12 (7), 569-571 (2005).</li><li>Meng, Y., Shao, C., Wang, H., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+all+the+miRBase-registered+microRNAs+true?+A+structure-+and+expression-based+re-examination+in+plants.">Are all the miRBase-registered microRNAs true? A structure- and expression-based re-examination in plants.</a> RNA Biology. 9 (3), 249-253 (2012).</li><li>Berezikov, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+flux+of+canonical+microRNAs+and+mirtrons+in+Drosophila.">Evolutionary flux of canonical microRNAs and mirtrons in Drosophila.</a> Nature Genetics. 42 (1), 6-9 (2010).</li></ol>

Our miRNA pipeline, SUmir, has been used for the identification of many plant miRNAs for the last decade. Here, we developed a new, fully automated, and freely available miRNA identification and annotation pipeline, mirMachine. Furthermore, a number of miRNA identification pipelines including, but not limited to the previous pipeline, were dependent on UNAfold software21, which became a commercial software over time, although once being freely available. This new and fully automated mirMachine is ...

Advances in next generation sequencing technologies have widened the understanding of RNA structures and regulatory elements, revealing functionally important non-coding RNAs (ncRNAs). Among different types of ncRNAs, microRNAs (miRNAs) constitute a fundamental regulatory class of small RNAs with a length between 19 and 24 nucleotides in plants1,2. Since the discovery of the first miRNA in the nematode Caenorhabditis elegans3, the presence and the functions of miRNAs have been studied extensively in animal and plant genomes as well4,5,6. miRNAs function by targeting mRNAs for cleavage or translational repression7. Accumulating evidence has also shown that miRNAs are involved in a wide range of biological processes in plants including growth and development8, self-biogenesis9, and several biotic and abiotic stress responses10.
In plants, miRNAs are initially processed from long primary transcripts called pri-miRNAs11. These pri-miRNAs generated by RNA polymerase II inside the nucleus are long transcripts forming an imperfect fold-back structure12. The pri-miRNAs later undergo a cleavage process to produce endogenous single-stranded (ss) hairpin precursors of miRNAs called pre-miRNAs11. The pre-miRNA forms a hairpin-like structure wherein a single strand folds into a double-stranded structure to excise an miRNA duplex (miRNA/miRNA*)13. Dicer-like protein cuts both strands of the miRNA/miRNA* duplex, leaving 2-nucleotide 3&#39;-overhangs14,15. The miRNA duplex is methylated inside the nucleus, which protects the 3&#39;-end of the miRNA from degradation and uridylation activity16,17. A helicase unwinds the methylated miRNA duplex after export and exposes the mature miRNA to the RNA-induced silencing complex (RISC) in the cytosol18. One strand of the duplex is mature miRNA incorporated into RISC , whereas the other strand, miRNA*, is degraded. The miRNA-RISC complex binds to the target sequence leading to either mRNA degradation in case of full complementarity or translational repression in case of partial complementarity13.
Based on the expression and biogenesis features, guidelines for miRNA annotation have been described15,19. With the defined guidelines, Lucas and Budak developed the SUmir pipeline to perform a homology-based in silico miRNA identification in plants9. The SUmir pipeline was composed of two scripts: SUmirFind and SUmirFold. SUmirFind performs similarity searches against known miRNA datasets through National Center for Biotechnology Information (NCBI) Basic Local Alignment Search tool (BLAST) screening with modified parameters to include hits with only 2 or fewer mismatches and to avoid bias towards shorter hits (blastn-short -ungapped -penalty -1 -reward 1). SUmirFold evaluates the secondary structure of the putative miRNA sequences from BLAST20 results using UNAfold21. SUmirFold differentiates miRNAs from small interfering RNAs by the identification of the characteristics of hairpin structure. Moreover, it differentiates miRNAs from other ssRNAs such as tRNA and rRNA by the parameters, minimum fold energy index &#62; 0.67 and GC content of 24-71%. This pipeline has been recently updated by adding two additional steps to (i) increase sensitivity, (ii) increase annotation accuracy, and (iii) provide genomic distribution of the predicted miRNA genes22. Given the high conservation of plant miRNA sequences23, this pipeline was originally designed for homology-based miRNA prediction. Novel miRNAs, however, could not be accurately identified with this bioinformatics analysis as it heavily relied on sequence conservation of miRNAs between closely related species.
This paper presents a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately (for example, the pipeline now uses sRNA-seq-based novel miRNA predictions as well as homology-based miRNA identification) and 2) is fully automated and freely available. The outputs have also included the genomic distributions of the predicted miRNAs. mirMachine was tested for both homology-based and sRNA-seq-based predictions in wheat and Arabidopsis genomes. Although initially released as free software, UNAfold became a commercial software in the last decade. With this upgrade, the secondary structure prediction tool was switched from UNAfold to RNAfold so that mirMachine can be freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline (examples are provided at https://github.com/hbusra/mirMachine.git).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>https://www.ncbi.nlm.nih.gov/books/NBK279671/</td>
			<td></td>
			<td></td>
			<td>Blast+</td>
		</tr>
		<tr>
			<td>https://github.com/hbusra/mirMachine.git</td>
			<td></td>
			<td></td>
			<td>mirMachine submission script</td>
		</tr>
		<tr>
			<td>https://www.perl.org/get.html</td>
			<td></td>
			<td></td>
			<td>Perl</td>
		</tr>
		<tr>
			<td>https://www.tbi.univie.ac.at/RNA/</td>
			<td></td>
			<td></td>
			<td>RNAfold</td>
		</tr>
		<tr>
			<td>Arabidopsis TAIR10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triticum aestivum (wheat, IWGSC RefSeq v2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mirmachine: a one-stop shop for plant mirna annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

The miRNA pipeline, mirMachine, described above was applied to the test data for the fast evaluation of the performance of the pipeline. Only the high-confidence plant miRNAs deposited at miRBase v22.1 were screened against the chromosome 5A of IWGSC wheat RefSeq genome v224. mirMachine_find returned 312 hits for the nonredundant list of 189 high-confidence miRNAs with a maximum of 1 mismatch allowed (Table 1). mirMachine_fold classified 49 of them as putative miRNAs depending on ...

Watch this Scientific Journal Video about mirMachine: A One-Stop Shop for Plant miRNA Annotation at JoVE.com

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

Computational identification of micro RNA's yields high false positive predictions. With the recent updates mirMachine provides high sensitivity and specificity in known and novel micro RNA predictions. MirMachine is proved to be superior to the most recent micro RNA algorithms in terms of sensitivity and mirMachine is now fully automated and freely available so everyone can use it with instructions provided.MirMachine can predict the genome-wide distribution of the putative micro RNAs without limitation of the data specific to tissues and conditions. Availability of the preliminary data prior to msRNA sequencing experiment would speed up the verification process for the important micro RNA genes. Before setting up the machine download and install software dependencies from the respective home sites using the links in the text manuscript.Alternatively, an easier and faster way to install the software dependencies is to use conduct using the commands provided in the text manuscript. Download the latest version of the mirMachine scripts the mere machine submission script from GitHub. Then set the scripts path into the path tab.Make sure that the mirMachine and its dependencies have been downloaded correctly by running the mirMachine on test data from GitHub. Check the job status on the screen by the standard output stream. Once all finished text appears, control the output files.Control the hairpins dot TBL dot out dot TBL output files. Notice the mature miRNA pre miRNA and the miRNAs star sequences on the second, third, and sixth columns. Find the location of the predicted miRNAs on the chromosomes at the end of each line.Then check the log files for the program outputs and warnings. For homology based identification run the mirMachine using the bash script and check the predicted mRNAs. Find the output file for the mature miRNAs and pre miRNA fastest sequences, as well as the output file for the hairpin log file.For novel miRNA identification pre-process the sRNA-seq Fast Q files into the proper FASTA format. Trim the adapters and remove low quality reads like those containing N as pre-processing for the sRNA-seq reads are not part of mirMachine. Choose a stable trimming tool and trimming parameters for the data submitted.Convert the FASTQ file into FASTA file as input file. If an abundance table file is provided use the modification script provided with the mirMachine scripts to convert the table file into a proper FASTA input. Then run the mirMachine using the bash script.Check the predicted miRNAs. Find the output file for the predicted mRNAs and prem miRNA FASTA sequences and the output file for the hairpin log file. Set the dash DB option to a blast database to skip the building reference database within the pipeline.Set the dash M option to the number of mismatches allowed and the dash N to the number of hits to eliminate after alignment. Change this based on the species. Use the dash long to assess the secondary structures for the suspect list, and the dash as to activate the novel miRNA prediction based on sRNA-seq data.Set the dash L max option to the maximum length of the S RNA-seq reads to include in the screening and the dash L min option to the minimum length of the sRNA-seq reads to have in the screening. Use the dash RPM option to set the reads per million threshold. In the representative analysis.The distribution of mRNA families from the chromosome five A of the IWGSC Wheat is displayed. The highest represented group of mRNAs was miR9666 with 18 identified miRNAs. Performance of the mirMachine on Arabidopsis thaliana and wheat species compared with the miRDP2.Comparisons of the sensitivity and the number two positives showed that mirMachine outperformed miRDP2 on the Arabidopsis data. For the wheat data mirMachine homology based prediction with expression evidence provided better sensitivity than miRDP2. For both genomes miRDP2 predicted a higher number of true positives than sRNAs-seq and homology based predictions.Setting up the required application could be burdensome for the inexperienced user. Following this video tutorial would help. Visualization of the installation process will encourage the non computational researchers to get into some basic computing.Additionally, investigating the output files in the video will definitely help the users interpret their results.

Research

JoVE Journal

Biology

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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

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A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

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High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...