2012 Annual Report
1a.Objectives (from AD-416):
1. An atlas of small RNAs will be created; 2. A website will be developed and publicly released for the visualization of small RNAs; 3. MicroRNAs will be identified, isolated, and evaluated; 4. Strand-specific, cleaved mRNA libraries will be used to identify targets of soybean microarrays.
1b.Approach (from AD-416):
Small RNA libraries will be constructed from tissues coordinated with ARS. Small RNA libraries will be generated in the University of Delaware lab using the improved Illumina TruSeq small RNA sample preparation kits, which eliminate all column purification and gel selection steps, are less time-intensive and more robust. These kits allow for decreased inputs of RNA; 500 ng total RNA is sufficient for library construction. Libraries will be bar-coded and four samples pooled and analyzed in a single lane on an Illumina HiSeq 2000 machine to yield single-end 50 base reads. We will quantitatively dissect the small RNA profiles across (a) tissues and (b) under stress treatments. Our proposed study will sequence far more deeply, with replicates, in a large array of tissues to give a comprehensive analysis of miRNA expression profiles in developing soybean tissues. By examining family member-specific Parallel Analysis of RNA Ends (PARE) data, we will distinguish expression patterns between members of highly duplicated, conserved miRNA families. PARE data is semi-quantitative but any interesting expression differences will be validated by precursor-specific RT-PCR. Between the analysis of these conserved families and the many novel miRNAs, we will generate extensive data on miRNA tissue specificity, and these will be validated as described below. Analysis of differentially expressed siRNA clusters may identify loci subject to epigenetic regulation during development or stress, which is as yet poorly characterized in plants. The deep sequencing of matched PARE libraries will define small RNA targets and greatly enhance the information gained from other small RNA and RNA-seq projects. The deep sequencing of PARE libraries allows a systematic experimental analysis of small RNA targets. PARE is based on a modified 5'-RACE and generates libraries containing 3' cleavage products of mRNA, including those caused by small RNA-mediated cleavage. Computational tools are then used to compare PARE signatures to small RNA data to identify miRNA-target RNA pairs (see Aim 2). In addition to validating predicted targets, PARE signatures provide an important resource to validate new miRNAs. A database containing PARE signatures from diverse tissue samples will therefore be a tremendously valuable resource for the soybean community that will enhance the information gained from all small RNA sequencing data. PARE libraries will be generated from the same tissue samples indicated above, but because of the difficulty in generating these libraries, we will be able to make only a quarter as many PARE libraries as small RNA libraries.
The first round of small RNA libraries was created from total RNA collected from open flower, flower bud, ovary and anther. The small RNA libraries for those samples were constructed and sequenced on the HiSeq 2000 at the Delaware Biotechnology Institute (DBI). For the existing libraries, we are currently running computational analysis on these data sets in order to identify novel miRNAs and phased, secondary siRNAs. We continue to advance and improve sample-handling infrastructure, focusing on tools for handling the data after they are submitted, meta-data handling, data pre-processing scripts, database building scripts, and visualization tools. We are integrating into our websites the set of soybean miRNAs that we had previously submitted to miRBase, the repository for known microRNAs. We have the algorithms optimized, and based on the first small RNA library data set generated from reproductive tissues, we are analyzing the sequencing data in order to identify miRNAs and as well as other classes of interesting small RNAs (i.e. phased siRNAs). We will continue and repeat these analyses as we accumulate more data to do the analysis. We have completed the development and testing of work to multiplex the Parallel Analysis of RNA Ends (PARE) library sequencing, allowing us to better perform quality control on these libraries. These modifications were tested by preparation of a set of libraries and as mentioned in the previous report, these have high complexity, and optimized for multiplexing of samples on the Illumina machine; we’ve now moved these protocols into production mode. The characterization of small RNAs in soybean will aid in our understanding of gene regulation.