Understanding the role of non-coding RNA
Small noncoding RNAs are involved in gene silencing and post-transcriptional regulation of gene expression. Deep sequencing of entire population of expressed small RNAs in a a given sample simuntaneously queries thousands of small RNA and miRNA sequences with extraordinary sensitivity and dynamic range giving insight in how post-transcriptional regulation contributes to phenotype. In addition, smallRNA-seq can be efficiently used for virus identification and discovery. The approach is highly sensitive and detects RNA viruses, DNA viruses and viroids that can be missed using targeted detection methods, such as ELISA or PCR-based tests.
Library preparation and sequencing
Our workflow can start with isolated total RNA or enriched small RNA. Upon the quality control of the starting material we generate libraries and select size in a range that maximizes the number of relevant reads. Sequencing is performed on NextSeq500 in a 75 bp single-read mode. Our establisehd protocol has provided successful library generation also on limiting templates and cumbersome samples (e.g. serum, exosomes, tree plants).
Standard bioinformatics analysis includes:
- Base calling and demultiplexing
- Trimming (removing lower quality bases and adapters)
- Count occurrence of each unique sequence (tag)
- Size distribution of tags creating a histogram of sequence size. The graph gives a first summary statistics on size distribution of the small RNAs sequences produced by the sequencing run
- Classification of tags and identification of noncoding RNA types
- Exact matching and counting of each miRNA and piRNA in a given sample - mapping reads against miRBase and piRNA entry
- Pair-wise differential expression analysis to identify known and novel microRNAs that are differentially expressed between sample groups
- Principal Component Analysis (PCA plot)
Advanced bioinformatics analysis includes:
- Virus identification and discovery
For every project we want to make sure that the outcome will meet your expectations.
If required we can perform RNA isolation from a range of samples including cells, tissues and biofluids.
We also provide full support on study design to ensure correct sequencing and bioinformatics strategies are used to meet your project goals. Our expert will consult with you about your specific requirements.