Background The use of new, deep sequencing technologies has greatly accelerated
October 9, 2017
Background The use of new, deep sequencing technologies has greatly accelerated microRNA discovery. have confirmed the expression of many microRNAs identified by sequence similarity and identified a pool of candidate novel microRNAs. Background MicroRNAs are small (about 22 nt) RNAs that play important regulatory roles by targeting mRNAs for degradation or translational repression. MicroRNAs were first identified in Caenorhabditis elegans  Rabbit Polyclonal to GPRC5B but high evolutionary conservation eventually led to the identification of microRNAs in other species. This, coupled with conventional sequencing of small RNA libraries, has greatly expanded the list of known microRNAs. The most recent release of the microRNA database, miRBase 10.0 , contains over 5000 microRNA gene loci in a wide variety of animal, plant and viral genomes. Conventional sequencing favors identification of highly expressed species, and comparative genomics will not identify nonconserved microRNAs. In order to enhance discovery of small RNA species, massively parallel signature sequencing (MPSS) was used to identify small RNAs in Arabidopsis thaliana , and the results showed that the diversity of small RNAs exceeded previous estimates. More recently, newer deep sequencing technologies have been used to profile microRNAs in Arabidopsis DICER and RDR2 mutants [4,5], and others have applied this technology to various samples including human and chimpanzee brain  and Chlamydomonas reinhardtii . These approaches have the advantage that they not only provide sequence of low abundance species, but also provide quantitative data since the frequency of sequencing reads reflects the abundance of microRNAs in the population. We previously reported on the use of deep sequencing technologies for identification of microRNAs encoded by Marek’s disease virus (MDV), an economically important pathogenic herpesvirus of chickens [8,9]. In an extension of the pilot study, we sequenced additional reads from both MDV-infected chicken embryo fibroblasts (CEF) and uninfected CEF and now report on the SB 431542 IC50 identification of potential novel host microRNAs. In addition, the sequence of several new MDV-encoded microRNAs were discovered by deeper sequencing. Results Small RNA libraries We obtained 256,221 reads from two small RNA libraries prepared from uninfected CEF or CEF infected with MDV. As shown in Table ?Table1,1, a total of 171,783 reads contained both adapters used in creating the library, and 125,463 of these high quality reads showed an exact match to the chicken genome. A total of 1 1,036 reads from the MDV-infected CEF library matched the MDV genome. The presence of other small RNAs (ribosomal fragments, tRNA, snRNA, mtRNA) was relatively small (less than 3%). Table 1 Distribution of small RNAs from uninfected CEF and CEF infected with MDV The majority (86%) of the small RNAs match to known or predicted chicken microRNAs (Additional File 1). Of the 149 distinct Gallus gallus (gga) entries in miRbase, we found 101 distinct species expressed in CEF. There were 93 matches from the MDV-infected CEF library and 87 matches from the uninfected CEF library. The infected cells showed slightly more complexity in microRNA diversity, which may be in part due to the larger number of reads obtained from the infected CEF library which increases the chances of revealing low abundance microRNAs. There were 12 microRNAs in the infected cells that were not found in the uninfected CEFs and 9 microRNAs found in the uninfected CEFs that were not found in the infected cells. An additional eleven chicken homologs of known microRNAs were identified (Additional File 1). SB 431542 IC50 The size distribution of reads was not significantly different in the two libraries, and the majority of the reads had lengths of 21C25 nt (Figure ?(Figure11). Figure 1 Size distribution of small RNAs. microRNA profiling by analysis of read counts The number of reads obtained should reflect the relative abundance and expression levels of the microRNAs. After scaling for total number of reads obtained for each library, the majority of microRNAs were found at similar levels in the two libraries. A few microRNAs (listed in Table ?Table2)2) showed a greater than two-fold difference in the number of reads between the infected and uninfected CEF libraries. We found miR-29b and miR-196 at higher levels in the MDV-infected cells, and three SB 431542 IC50 of the let7 microRNAs were found at lower levels in the MDV-infected CEF compared to the uninfected CEF. Northern blot analysis didn’t detect these distinctions, but this may be due to the.