This tool predicts sRNA loci using the method described in [1] and [2]. It also enables the user to compare the expression profile of sRNA loci between difererent samples. In order to determine the relative position of sRNAs, the reads are mapped to the reference genome using PatMaN [3]. Only full-length, perfect matches are accepted as hits. The genome-matching reads are normalised [4] and weighted by repetitiveness. The normalisation method divides hit counts by the number of redundant reads that match the genome. The normalised count, for each distinct read, is given in \hits per 1 million matching reads”. Because it is impossible to decide where a sRNA with multiple matches to the genome originated, we correct the normalised read-abundance for repet- itiveness by dividing it by the number of matches to the genome. The result is a weighted hit count. The method uses the normalised and weighted read-abundance and relative position of sRNAs on the reference genome to predict the sRNA loci. A locus must have a minimum of 3 weighted sRNA hits (this threshold can be adjusted using the min hits parameter) and no gap (absence of sRNA hits) longer than 300nt (this threshold can be adjusted using the sRNA loci distance parameter).

The datasets must contain sRNA sequence reads in FASTA format, in redundant form, i.e. with one entry for each read. Sequences shorter than 18nt (minsize parameter) or longer than 30nt (maxsize parameter) will be removed.

Required Parameters:

  • Genome File: The location of the genome file in FASTA format.
  • sample names: The locations of the sRNA samples

Input files are entered using the box displayed below:

Input dialogue for SiLoCo. Enter your databases for each sample and your genome file

Optional Parameters

  • sRNA loci distance: The maximum gap length in a locus, default max gap = 300).
  • max size: The maximum length of a sRNA.(18 maxsize 35, default maxsize = 25).
  • min size: The minimum length of a sRNA.(18 minsize 35, default minsize = 25).
  • min sRNA locus size: The minimum number of sRNAs in a locus.(1 min hits, default min hits = 3).
  • max genome hits: The maximum number of times a sRNA can hit the genome.(18 minsize 35, default minsize = 18).

The results are presented in a Table as shown in the image below.

An example of the results from a two sample SiLoCo run

The headers for each column contains the description of the data and the name of the sample file. Locus-data is shown in a table with the following columns:

  • Chromosome, start/end position and length Genomic location and length of locus in nucleotides. Some incomplete genomes may not yet be assembled into chromosomes and the acces- sions listed here may be scaffolds or bacs instead. The list is initially sorted by chromosome and position.
  • Raw count Sum of read abundances in samples 1 and 2 that from the locus (not corrected for repetitiveness).
  • Weighted count Sum of raw read abundances divided by number of matches of each sequence to the genome.
  • Normalised count Sum of weighted counts divided by the total number of genome-matching reads in each sample, given in \hits per 1 million genome-matching reads. Normalised counts (abundances) are comparable between sam- ples.
  • Uniquely matching reads (optional) Number of sequence reads in the locus that only have a single match to the genome.

The context menu operates on the currently selected result line. ‘Show in VisSR’ will display the selected locus in VisSR. An example of a locus shown in VisSR is below:

siloco in VisSR
A locus, predicted in SiLoCo and displayed in VisSR

[1] Attila Molnar, Frank Schwach, David J Studholme, Eva C Thuene- mann, and David C Baulcombe. mirnas control gene expression in the single-cell alga chlamydomonas reinhardtii. Nature, 447(7148):1126 1129, Jun 2007 [2] Rebecca A Mosher, Frank Schwach, David Studholme, and David C Baulcombe. Polivb induences rna-directed dna methylation indepen- dently of its role in sirna biogenesis. Proc Natl Acad Sci U S A, 105(8):31453150, Feb 2008 [3] Kay Prufer, Udo Stenzel, Michael Dannemann, Richard E Green, Michael Lachmann, and Janet Kelso. Patman: rapid alignment of short sequences to large databases. Bioinformatics, 24(13):1530{1531, Jul 2008 [4] Ali Mortazavi, Brian A Williams, Kenneth McCue, Lorian Schaevter, and Barbara Wold. Mapping and quantifying mammalian transcrip- tomes by rna-seq. Nat Methods, 5(7):621:628, Jul 2008

A suite of tools for analysing micro RNA and other small RNA data from High-Throughput Sequencing devices