respectively). A total of 14,034,295 raw sequences of small RNAs were obtained by Solexa deep sequencing (Illumina). After alignment to the mouse genome (mm9), the resulting total number of genome-aligned reads was 11,181,999 (80%). The number of sequence reads that correspond to known miRNAs – 7,049,494 (63% of genome-matching reads) – was determined by perfect sequence matching to the database of known miRNAs (miRBase release 16). We checked for the expression of 1055 different mouse miRNA types annotated in miRBase (release 16) [19] – these include star sequences as well as alternate miRNA products (denoted as 25p/23p or, sometimes, -s and -as). We detected perfect matches to 558 of these miRNA products in our mouse brain samples (Table S1). To compare the differential expression of miRNAs in the brain of young mice versus old mice, the number of miRNAs in each sample was normalized to the total number of reads (sequencing depth) in each sample that matched the mouse genome (mm9) (old/young = 1.472107). At least 184 miRNAs were considered to be differentially expressed (P-value ,0.05, as determined by DEGseq [20]) (Table S2). To generate a conservative list, we filtered this data set by considering only miRNAs with read counts $10 in at least one time point sample (miRNAs with fewer reads did not exhibit dramatic expression changes). This resulted in 172 miRNAs (bolded in Table S2). An additional 108 sequences were previously annotated as miRNA star sequences and are reported separately (Table S3). Candidates that we initially identified in our novel miRNA discovery pipeline (see below) but were subsequently reported in the latest release of miRBase (release 16) were also added to the list of differentially expressed known miRNAs. Novel miRNA Discovery To uncover novel miRNAs, we used the software miRDeep, which was developed to identify novel miRNAs in deep sequencing data [21]. The algorithm takes into account several factors to score the probability that a candidate small RNA found by deep sequencing represents a bona fide miRNA: the presence in the deep sequencing data of reads corresponding to typical products of miRNA biogenesis; the stability of the putative pre- miRNA hairpin; and homology to previously identified miRNAs. Beginning with 11,181,999 genome-matching reads, and after discarding reads corresponding to previously annotated regions, miRDeep analysis revealed the existence of a total of 41 novel candidate miRNAs (Figure 1(a), Table S4). Some of these novel miRNA candidates share seed sequences with known miRNAs in mouse and other species (Figure 1(a), (d)), as determined by the miRDeep algorithm or through BLAST sequence alignment, providing additional evidence that they are bona fide miRNAs. Interestingly, the mature sequence of one novel miRNA candidate, seq_96718_x14, is nearly identical to miR- 378-3p (one nucleotide difference), but alignment of the precursor sequence mapped seq_96718_x14 to chromosome 10 rather than to chromosome 18 on which miR-378 resides. These precursor sequences share little conservation. Thus, while seq_96718_x14 may be a mir-378 family member, it is distinct from miR-378-3p and not merely a single nucleotide variant occurring from the same locus. Indeed the differing nucleotide falls within the seed region (Figure 1(d)), suggesting that the two miRNAs regulate different sets of genes. One miRNA matched perfectly with a miRNA in another species, suggesting it to be a mouse homolog. Novel miRNA candidate seq_169464_x8 aligned perfectly with rno-miR-3552; the genomic contexts of the two miRNAs are also conserved. It is important to note that some novel miRNA candidates were mapped to genomic regions overlapping known Novel MicroRNAs in the Aging Mouse Brain miRNA gene sequences, but the mature sequences were distinct and therefore considered distinguishable from the overlapping known miRNAs. These miRNAs are referred to as alternative forms (isomiRs) of the known miRNAs in this paper (miR-341 isomiR, miR-344-1 isomiR, and miR-5620 isomiR). Other miRNA candidates did not share sequences with any known miRNAs and may represent a group of miRNAs expressed only in aging- or brain-specific contexts. Three novel miRNA candidates overlapped with small nucleolar RNA (snoRNA) sequences (miR- 6516, seq_692265_x97, and seq_93483_x4), and two more mapped to several genomic loci when BLASTed including ribosomal RNA (rRNA) sequences (seq_328941_x27 and seq_321717_x6). Validation of Candidate Novel miRNAs We next evaluated the likelihood that novel miRNA candidates represent true miRNAs. The secondary structures of putative pre- miRNA hairpins were generated using RNAfold [22]; structures of select novel miRNA candidate precursor sequences are shown in Figure 1(b). Although demonstrating differing complexities, all novel miRNA candidate precursor sequences fold into hairpin structures characteristic of miRNA precursors. With respect to genomic locations, novel miRNA candidates are found in exonic, intronic, and intergenic regions, with no apparent bias. Finally, we tested the expression of nine novel miRNA candidates via TaqMan qRT-PCR and confirmed the expression of 8 (Figure 1(c)). miR-5620 (isomiR) was detected but with late Cp call. Mapping Novel miRNAs to Genomic Locations We examined the genomic locations of brain-expressed miRNAs to see if miRNAs found in particular genomic regions were potentially coexpressed and, therefore, potentially coregu- lated. Clusters of known miRNAs were retrieved from the miRBase website. Many miRNAs are located within close proximity (,10 kb) on chromosomes 12 and X suggesting that these miRNAs might be coexpressed (Figure 2). Several novel miRNA candidates also were found in close proximity to these clusters. Some of these miRNAs mapped to the same transcript. It may be important to note, however, that not all known miRNAs lying within 10 kb of brain-expressed miRNAs were expressed in our data sets, thereby suggesting distinct expression regulatory mechanisms for these miRNAs. Dynamic Expression of miRNAs in Brain Aging Many miRNAs expressed in the brain demonstrated dynamic expression changes with age (Figure 3(a)). 18 novel miRNA candidates and 75 known miRNAs demonstrated $2.0-fold differential expression with age (Figures 1(a) and 3(b)) (known: 4 upregulated, 71 downregulated; novel: 4 upregulated, 14 downregulated). While specific miRNAs were upregulated in expression with increased age, most of these differentially expressed miRNAs demonstrated decreased expression, in agree- ment with trends reported in previous studies that profiled miRNA expression changes with age [12,18]. It is interesting to note that, on average, novel miRNA candidates were detected at copy numbers roughly 3 orders of magnitude lower than that of known miRNAs, providing one explanation as to why they might not have been previously identified. A few known miRNAs were selected for validation by qRT-PCR (Figure 3(c)). MiR-124 and miR-34a showed similar expression trends in qPCR and deep sequencing data, but we were not able to detect large changes in expression of miR-17 with age using qPCR. Deep sequencing and qPCR differ on sequence end recognition and are not always PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40028