sRNAbench

Profiling of small RNAs and their sequence variants in single or multi-species high-throughput experiments

Description

Several commonly used input formats like fastq, sra, fasta or read/count are supported. Input files can be provided via upload, URL, SRA accession, Google Drive or Dropbox. sRNAbench performs expression profiling of known microRNAs and other types of small RNAs, detection and analysis of isomiRs, outputs several graphical summaries and predicts novel microRNAs in both plants and animals. Result example

Sequencing data input page

The sequencing data can be provided in 5 different ways:

• Upload one or several files (typically fastq or fastq.gz)
• Provide one or several link/URL(s) with the data (it will be downloaded and then profiled)
• Provide one/several accession(s) for SRA runs (they start with SRR, ERR or DRR e.g. SRR1563062)
• Choose files from your Google Drive account
• Choose files from your Dropbox account

To add samples, simply click on the desired button and then add the files using the prompt or add the SRA accessions/URLs (one per line) and click on the “ADD” button. Once all your read files have been provided, you can click “Next” to move on to the parameter specification. Traditionally, we have supported an unlimited number of samples to be uploaded but we may revisit this policy if some users start engaging in abuse of these conditions.

Example on how to use SRA data

The following link will take us to the SRA project page which contains all information provided by the authors (see below).

This table can be downloaded and the SRR identifiers can be extracted and pasted into the corresponding textbox (see below).

Input parameters page

1. Select species annotation:

Alignment of small RNA reads requires at least a miRNA reference annotation or genomic annotations. The left side dropdown selector allows you to choose the miRNA reference database (MirGeneDB or PmiREN are recommended if available), the right side selector allows you to choose one or several species of interest. Then the remaining selector will let you choose genome assembly and annotations from Ensembl for one or several species of interest. You can decide not to map to the genome (slightly faster but miRNA/sRNA loci cannot always be distinguished), not to profile to other ncRNAs or if you want to predict novel miRNAs from your sequencing data (which requires a genome assembly). This prediction will be performed after all miRNA and other ncRNA reads have been assigned.

2.Reads preprocessing:

We have implemented five different library preparation protocols for straightforward adapter removal. If your experiment was not designed using these five protocols, custom settings can still be selected. If reads are already trimmed, it can also be specified here.

Optional parameters: - Quality control: There are two methods to filter out reads of low quality: mean-based, a read is accepted if the mean Phred score is above a given threshold; and minimum per nucleotide, a read is eliminated if a single position of the read has a Phred score below the given threshold. The Phred threshold can be defined by the user. - Parameters: Other parameters that can be adjusted like bowtie mapping parameters and minimum read counts or maximum and minimum read lengths.

Status page

Samples launched will be listed in the status page. In this table users can check information about the status of their job, start and finish time, what input was used and name or group information (if provided) as well as a link to navigate to the individual result of each sample. To provide sample names and groups for differential expression (once all jobs are done running) you can click on the button “Annotate Samples”. This will take you to a different page where samples can be annotated using an excel file. Other functions include bulk zipped download of all results, job relaunching (sample samples with different parameters), Differential Expression (Launch sRNAde) and Matrix Generation.

• Launch jobs on SRNAde: Once all sRNAbench jobs are finished aligning, the user can launch an sRNAde job to perform differential expression. Users can navigate to a new page where they can manually provide groups/conditions for their samples or they can use annotations previously provided using the “Annotate Samples” button.
• Annotate samples:

Users can easily annotate their samples using this feature which provides a pregenerated template (Download template file) with their samples and sample names or sample groups if they had previously been annotated. If a sample is not assigned to a group (i.e. left blank) it will not be taken into consideration for the differential expression analysis.

• Generate matrix:

By clicking on the “Generate matrix” button users can access this menu where different files from sRNAbench can be summarized into a matrix. For some matrices, a visualization is also available.

Individual job output page

By clicking on the link of each sRNAbench job, users can access a report for each individual sample. There are five sections in the sRNAbench report:

• Parameters: a display of all the parameters used to run the job including sequencing protocol, genome/species and input file.
• Summary: a series of preprocessing and mapping statistics/plots that can provide an overview of the quality of the sequencing library and of the mapping.
• Genome mapping: a summary of the reads mapping to genome and unique reads.
• microRNA summary: a summary of reads mapping to miRNAs (selected reference and genomes) including plots and links to tables to individually explore each mature miRNA sequence, hairpin align files and isomiR distribution.
• sRNA summary: similarly to the previous section, abundance tables are provided for each sRNA category both using single and multiple assignment.
• Other features: this tab is designed to contain current and future functionalities that do not fall within any previous section. Right now, it contains anticodon statistics of tRNAs present in the profile.

Besides these sections, users can download a zip file containing all the result files of the sRNAbench command line tool (check the tool manual for more details). Feel free to contact us if you cannot find a file you were expecting in there or if there is some information you would like to see included.