Small RNA Sequencing Bioinformatics Capabilities

Seven point small
RNA quality filtering

  • Trim 3' adapter from termini
  • Base quality filter
  • Remove reads <17 nt after trimming
  • Remove untrimmed reads
  • Remove reads containing 5' adapter
  • Remove reads with unknown nucleotides
  • Remove low-abundance reads

ORB maintains a well-established, thoroughly vetted, and highly parallelized computational pipeline for rapid and cost-effective processing of small RNA sequencing data from Illumina sequencing runs. Pre-processing small RNA sequencing reads is critical to achieve reliable mapping and quantification; thus ORB utilizes an 7-point quality filter on small RNA sequencing reads to ensure efficient and precise mapping to small RNA databases. Trimmed sequence reads that have passed all quality filters are aligned to genomic and mRNA sequences, and reads mapping to the genome are further mapped to 9 classes of small RNAs, including tRNA, rRNA, snoRNA, microRNA, microRNA precursor hairpins, piRNA, transcripts, retrotransposons, and YRNA.

A wide selection of customizable bioinformatics analysis services are offered to provide our clients with a comprehensive and all-inclusive package to support their data analysis requirements. Examples of customized small RNA analysis include prediction of novel microRNAs based on MirPara 6.2 software, target microRNA-gene pair prediction based on three databases including miRDB, TargetScan, and miRTarBase, and enriched pathway analysis of genes targeted by differentially expressed microRNAs. 


ORB’s Small RNA Sequencing Data Analysis Packages

Standard Analysis Package
  • Full sequencing QC report
  • Sequence read counts and alignment for known microRNAs
  • Fold Changes, 1 and 2 - way ANOVA, t-tests
  • Principal Component Analysis (PCA)
  • Hierarchical clustering
  • PowerPoint summary
  • Raw FASTQ files
Additional Analysis
  • Full statistical analysis
  • Alignments for other small RNAs - piRNA, snoRNA, snRNA
  • Sequence read counts, alignment and hierarchical clustering for novel microRNAs
  • MicroRNA target prediction pathway analysis
  • Linear and logistic regression
Custom Analysis Capabilities
  • Logistic regression
  • Non-parametric testing
  • Mixed-Effect models
  • Correlation of microRNA and mRNA sequencing data
  • Predictive modeling


ORB’s Small RNA Analysis Pipeline
  • Summary report provides unique sequence list with counts for all samples and list of known annotations.
  • Ensembl transcript database mapping enables investigation of 40 additional RNA biotypes.
  • Novel microRNA prediction based on peer-reviewed MirPara 6.2 software.
  • Analysis of read counts based on either perfect match or single-mismatch alignment filtering.


Novel miRNA Prediction Pipeline

ORB has developed a comprehensive workflow for predicting novel microRNAs from small RNA sequencing reads using MiRPara.

Novel microRNA Prediction Workflow

Small RNA Sample Data Set

Results package of a sample dataset is provided in .zip archive format; to download the package click on the PowerPoint slide image below.

"ORB Small RNA Seq Demo Analysis"


ORB’s small RNA sequencing services are frequently utilized in biomarker discovery programs to identify screening, prognostic, and diagnostic markers, particularly with biofluids to support the use of minimally invasive sample collection strategies in the clinical setting. The the co-development of companion diagnostics and therapeutic agents are often supported with small RNA characterization studies and subsequent validation studies with real-time qPCR assays, which can be GLP certified as needed. ORB has created a highly customizable predictive modeling system to identify reproducible and sensitive microRNA biomarkers. Follow the links below for additional details on these applications.