mirMachine: A One-Stop Shop for Plant miRNA Annotation

Summary

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.

Abstract

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.

Introduction

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 plants^1,2. 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 well^4,

Protocol

1. Software dependencies and installation

1. Install software dependencies from their home site or using conda.
   1. 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.
   2. 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 BLAST 2.6.0+.
   3. Install precompiled package of RNAfold from https://www.tbi.univie.ac.at/RNA/.
   4. Alternatively, install these softwa....

Results

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 .......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
https://www.ncbi.nlm.nih.gov/books/NBK279671/			Blast+
https://github.com/hbusra/mirMachine.git			mirMachine submission script
https://www.perl.org/get.html			Perl
https://www.tbi.univie.ac.at/RNA/			RNAfold
Arabidopsis TAIR10
Triticum aestivum (wheat, IWGSC RefSeq v2)