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The sRNAs obtained were subjected to a BlastN search against known non-coding RNAs deposited in the Rfam 11.0 and NCBI Genbank databases in order to enable the removal of tRNA, rRNA, snRNA, snoRNAs and scRNA sequences: these accounted for 5.32% of the reads in R1, 8.20% in R2, 4.66% in R3 and 5.64% in R4 (Table 1).
All 8 predicted miRNAs were classified into different microRNA families based on their precursor sequences in Rfam web server (Griffiths et al.
Library Tag counts Average length Tags annotated after removing of tags (nt) to miRBase low quality sequences S-M 4,949,795 29.5 258 S-PI 4,712,304 29.3 432 R-M 3,384,144 28.6 185 R-PI 3,021,557 28.4 368 Library Number of Number of Real precursor ESTs matched ESTs after miRNAs to annotated BLASTX and conformed tags Rfam by SVM searches S-M 3253 1178 42 S-PI 4750 2293 43 R-M 1730 694 30 R-PI 4021 1298 27 TABLE 2: Statistics of the characterized parameters of wheat miRNA precursors.
Tate et al., "Rfam: updates to the RNA families database," Nucleic Acids Research, vol.
These clean reads were aligned against Rfam database to identify the reads classification using Bowtie .
Sequences recognized as long non-coding RNAs (lncRNA) in the Rfam and NONCODE databases were further distinguished from transcript-coding proteins (Wu et al., 2012).
The plant pre-miRNAs from the microRNA Registry Database (Version Rfam 20 released June 2013) (Griffiths-Jones, 2004), and PMRD (Zhang et al., 2010) were downloaded and subjected to BLAST against publicly available 15,105 Prunusarmeniaca ESTs from the database, i.e., dbEST release 130101 at http://blast.ncbi.nlm.nih.gov/Blast.cgi using blastn (Altschul et al., 1990).
We parsed the mapping results to identify reads mapped to miRNAs (miRBase, release 19), piwi-interacting RNAs (piRNAs) (piRNABank, November 2013), and other known small RNAs (RFam, version 11.0).
We performed similarity searches through blastx against UniRef90 and Swiss-Prot (v201706, word_size = 4; e-value = 0.00001), rpsblast against the Conserved Domain Database (CDD) profiles (ftp://ftp.ncbi.nih.gov/pub/mmdb/ cdd/little_endian/Cdd_LE.tar.gz, e-value = 0.00001, num_descriptions = 20, and num_alignments = 20), and blastn against Rfam and rRNAs (e-value = 0.00001, num_descriptions=1, num_alignments= 1, and num_threads = 4).
The clean sequencing data were mapped into two genome and Rfam database v11 (http://www.sanger.ac.uk/Software/ Rfm/).
From remaining 11433100 reads total putative 4968 siRNAs were identified according to the criteria that these sequences do not derived from hairpin structures and are not related to other ncRNAs deposited in Rfam and NT databases.
Then, the clean reads were compared against the non-coding (ncRNAs) (rRNAs, tRNAs, snRNAs, and snoRNA) deposited in the NCBI GenBank and the Rfam databases using BLAST to annotate the small RNA sequences.
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- RFamide-related peptide receptor OT7T022