Small RNA Discovery & Profiling

Small RNA-Seq can be used to discover novel miRNAs and other small noncoding RNAs, characterize variations such as isomiRs with single-base resolution, and analyze any small RNA or miRNA without prior sequence or secondary structure information. Many library Prep Kits are there to supports customizable size selection to investigate any small RNA between 17 to 35 nucleotides and analyze the expression levels of both known and unknown miRNAs in your sample.

Eurofins genomics offers comprehensive small RNA sequencing on Illumina platforms. We offer customized experimental design of small RNA sequencing for plant, animal, insects etc. We have developed standard pipeline for bioinformatics analysis of small RNA sequencing data and provide support for end to end analysis.

Workflow and deliverables

a. Quality check and filtrating

In small RNA analysis pipeline, the other RNA contaminants such as rRNA, tRNA and adapter sequences will be filtered out from miRNA transcriptome data of the sample. Then, the filtered reads of the samples will be mapped on the miRBase precursor sequences.

b. Mapping to mature miRNA

Mature miRNAs will be focused for homology based search, rather than miRNA precursor sequences, as they are conserved in plants and animals. The filtered reads of the samples will be mapped individually against precursor miRNAs using GFF file.

c. Identification of mapped mature miRNAs

The co-ordinates of the mature miRNA sequences will be obtained from miRBase mature co-ordinates and GFF files. The expression analysis of mature miRNAs will be carried in the samples by computing FPKM values.

d. Differential expression of mature miRNAs

The FPKM value for each mature miRNA will be calculated for the samples. The FPKM values of common miRNAs will be used to calculate the log fold change as ln(FPKM_stress/FPKM_control) (where In is natural log).

e. Novel miRNA prediction

The small RNA reads which represent ten or more times in data will be considered for novel miRNA predictions. MiRanalyzer pipeline / miRDeep2 pipeline will be used to predict potential miRNA precursor molecules, by mapping them on reference genome.

f. miRNA targets prediction

Targets identification will be done using Targetscan/miRanda/PicTar/psRNATarget.

g. Functional annotation of target sequences

Functional annotation will be performed for the predicted target sequences against NR, COG, KEGG databases using BLAST.

Deliverables

Small RNA discovery

• Quality filtration of reads
• Identification of known miRNA
• Expression profiling of miRNA
• Differential miRNA analysis(if more than one sample)
• Novel precursor miRNAs and mature miRNA
• Secondary structures of candidate(novel) precursor miRNAs
• Target identification of predicted and differentially expressed miRNA,
• Target Gene ontology (if available)
• Target Pathway (if available)
• Comprehensive report with publication standard methodology, graphs and tables.