differences among the miRNAs. Therefore, it may be difficult to determine whether the sequences that match these miRNAs are sequence error or whether they are isomR. For example, porcine F37976_x4, F1_x405541, and F500_x373 from the Solexa sequences matched dya-miR-1, ptr-miR-1, and rno-miR-1, respectively. These three miRNAs have sequence differences, so F37976_x4, F1_x405541, and F500_x373 may be isomiR of miR- 1 in the pig. However, miRNA end nucleotides are difficult to identify, irrespective of whether miRDeep or homolog search is used. This is especially true for the 59 end sequences, which are important because they contain the ‘‘seed’’ region of the miRNAs. Taken together, miRDeep can provide accurate annotations of miRNAs from short sequences. When the detected miRNA from miRDeep has a homolog in the model species, the end nucleotides can be determined according to the homologous miRNAs. To find other porcine miRNAs which may not have been captured by using Solexa sequencing, miRDeep program was re-adopted to predicate miRNA in porcine genome (method3), the main advantage of using this method is that some tissues specific and/ or developmental specific miRNAs, which not detected by Solexa sequencing may be found in silico method. Analyses of the 437 conserved and 86 candidate novel miRNAs identified by different methods showed that 116 miRNAs are 21 nt long (22.2%), 212 miRNAs are 22 nt long (40.5%), and 99 are 23 nt long (18.9%). This is in agreement with the library sequence distribution analysis results, which showed that sequenc- es that were 21–23-nt in length had the highest frequency. A total of 66 piwiRNAs were obtained by homology search (Table S11). Some rRNA, tRNA, and sno/snRNAs were also found in the library. The proportion of miRNAs was 5.64% in all of the nonredundant sequences in the library (Table S14). The percentage of miRNAs was 65.12% when the redundant sequences were counted. Most of the sequences (91.01%) had more than 1 read (Table S15). Some sequences could not be annotated, which may be due to the incomplete nature of the pig genomic sequence. To show the sequence variations between our library and 212 porcine miRNAs in miRBase (Release: 16.0) [24,25,26,27], we found that 115 miRNAs identified from this study had perfect match with each other, and 77 had 1 or 4 base variations at the length of terminus. Among the sequence identical group, we found a candidate novel miRNA miR-new66 had perfect match with the known porcine miRNA ssc-miR-676-3p (miRBase 16.0). More- over, two of the 86 candidate novel pig-specific miRNAs were also identified in a porcine skeletal muscle library in our lab [30]. A total of 88 of the 296 miRNAs identified by Huang et al. (2008) had perfect matches in our library [28]. Using the Solexa sequencing and microarray techniques, the expression of 332 intestinal miRNAs was uncovered [34]. Comparison of these revealed that a total of 139 of the 332 miRNAs identified by Sharbati et al. (2010) had perfect matches in our library, including 8 novel pig- specific miRNAs, such as miR-new3, miR-new6, miR-new10, miR-new16, miR-new19, miR-new28, miR-new56, miR-new66. In addition, some of them were validated by the stem-loop RT- PCR method (Figure 6). These findings further support the notion that they may exist in the pig. While our manuscript was prepared, one paper was published on PLoS ONE in which hundreds of miRNAs were identified in the pig by Solexa sequence[40]. When we compared this data with ours, we found that although those researchers performed a relatively comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained in different ages, only 272 had perfect matches with the miRNAs identified in our study. There were 83 miRNAs had 1 or 2 base variations. Compared with all Discovery of Porcine MicroRNA by Solexa Sequencing the reported porcine miRNAs, we found that 230 miRNAs, including candidated novel miRNAs, which identified from this study had perfect matches with each other, and 120 had 1 or 2 base variations at the length of terminus. In other words, 112 conserved and 61 candidate novel porcine miRNAs were first reported in our study. We also compared the identities of porcine miRNAs from this study with known human, mouse, and rat miRNAs (miRBase 16.0). A total of 414 porcine miRNAs in our study matched (identity.95%) with their human, mouse, and rat counterparts. This result showed that porcine miRNAs had high homology to miRNAs from human and rodent, which was in agreement with previous reports [24]. Several studies showed that isomiRs may function in the animal [41,42]. Our analysis revealed a large number of isomiRs, derived from almost all detected miRNAs, in total, we detected 23822 isomiRs in our library, which greatly exceeded and extended previous reports. Further analysis revealed two major classes of variants or isomiRs(Table S10). Most isomiRs showed variability at their 59 and/or 39 ends, likely resulting from variations in the pre-miRNA secondary structures that result in variable cleavage sites for Dicer1 or Drosha. An example is given in Figure 3, demonstrating sequenced isomiRs for miR-1, which indicated that the majority of porcine miRNA nucleotide variants resulted from post-transcriptional modifications. The presence of isomiRs may have various functions and require further research. The seed sequence is formed by seven or eight nucleotides of the mature miRNA, such seed regions are mostly conserved among miRNAs of the same family [5]. Here we used 8mer sites (an exact match to positions 2–8 of the mature miRNA) as parameter to classify different miRNA families, especially the candidate novel miRNAs. As a result, all the mature miRNAs can be classified into 300 different miRNA families (Table S16). We further compared the largest conserved miRNA families among different species, the let-7/98 family. This family is the first miRNA family to be reported. There is a very low rate of nucleotide change among its members. The human let-7/98 family contains 9 members, i.e., hsa-let-7a/b/c/d/e/f/g/i and hsa-miR-98. The rat let-7/98 family has 8 members (let-7g is not included). We found that the porcine let-7/98 family has 12 members including the 9 human homologous and ssc-let-7h/j/k miRNAs (Table S16). By analysis of candidate novel miRNAs, we found some of them can be classified into the conserved miRNA families, for example, the seed sequence of miR-new42 is exactly the same as the miR-28 and miR-708’s.The 59 end sequences are crucial for classifying the miRNA families, and a change in even one base pair can result in failed classification of members of the same family. Based on these results, we concluded that our Solexa library is of high quality and allows easy identification of pig miRNAs. Using our porcine miRNA-array, 123 miRNAs were detected expression in porcine liver tissues, including 19 new miRNAs found by Solexa sequencing. Among the top 10 expressed miRNAs, miR-122 has been reported as an abundant liver- specific miRNA [43,44], other highly expressed miRNAs were firstly reported in this study. There were 58 miRNAs differentially expressed between the Tongcheng and Large White breeds. Let- 7a was the most highly expressed let-7 family member, it has been confirmed as a negative regulation signal transducer and activator of transcription 3 (STAT3), which is associated to hepatocellular proliferation and hepatocarcinogenesis [45]. TGFbeta-R1 also had been confirmed as a target gene of let-7c, which participated in liver development. These results indicated that let-7 family members play important roles in liver development [46]. Let-7 miRNAs were differentially expressed between Tongcheng and Large White pigs, indicating the gene expression regulation at PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16235