A new library manufacturing method for analyzing the miRNAs by massively parallel sequencing (miRNA-seq) has been developed.
By nanotechnology, it is possible avoiding the ligation reaction during the NGS library preparation, a common step in this process and the main source of bias. In this way, it allows reducing the appearance of these ligation biases and other associated problems.
MiRNAs are small RNA molecules involved in regulating the expression of numerous genes. The dysregulation of these miRNAs is associated with most of the human chronic pathological processes (cancer, diabetes and neurodegenerative diseases), so their analysis represents a field of increasing relevance. However, its small size and low concentration hinder the study and precise quantification of the miRNA species present in a biological sample.
At present, although parallel-massive sequencing, miRNA-seq, is postulated as the most promising technique for the analysis of these species, reproducibility problems associated with the library preparation stage make its application difficult.
The present invention proposes a method of manufacturing miRNA libraries for massive sequencing in the absence of the ligation reaction. Habitually, adapters add by this reaction that leads to the appearance of biases. These considerably alter the relative frequencies of the miRNA species. Our technology makes it possible to eliminate these binding biases, achieving an increase in efficiency and costs reduction.
The preparation of the libraries carries out using nanotechnology. In this way, the necessary massive sequencing adapters add by Taq polymerase, without requiring the ligation reaction. Therefore, it uses magnetic particles that have 5' covalently linked oligonucleotides on their surface, on which the miRNA is reverse transcribed and the sequencing adapters by Taq polymerase are added.
ADVANTAGES AND BENEFITS
- Reduction of biases.Ligation bias is an unsolved methodological problem whose error is difficult to quantify. The use of nanotechnology allows eliminating the ligation reactions of the process and, consequently, suppressing the biases associated with it.
- Increased efficiency.Our library manufacturing process achieves an increase in the efficiency of the process that is affected in the usual procedures by the problems associated with the development of the ligation reaction.
- Reproducibility of results.The manufacture of libraries in the absence of a ligation reaction makes it possible to solve reproducibility problems due to the biases caused by this reaction.
- Cost reduction.The lesser appearance of problems during the manufacturing process and the consequent improvement in efficiency promotes to reduce the associated costs.
Current development status
Desired business relationship