Systems and methods for making sequencing libraries
The method of random nucleic acid fragmentation and reverse transcription with enhancers addresses the limitation of existing methods by creating a library of uniquely identifiable polynucleotides with diverse cleavage sites and enhancers.
Patent Information
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- ILLUMINA INC
- Filing Date
- 2026-06-04
- Publication Date
- 2026-07-17
AI Technical Summary
Existing methods for creating libraries of molecularly distinct polynucleotides are limited in their ability to generate uniquely identifiable fragments with random cleavage sites and molecular diversity enhancers.
A method involving random fragmentation of nucleic acids followed by reverse transcription in the presence of molecular diversity enhancers to create polynucleotides with identifiable combinations of cleavage sites and enhancers.
Enables the generation of a library of polynucleotides that are uniquely identifiable based on random cleavage locations and molecular diversity enhancers, enhancing molecular diversity.
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Abstract
Description
Abstract This invention relates to systems and methods for creating libraries of molecularly distinct polynucleotides. In particular, the methods of the invention involve randomly fragmenting nucleic acids (e.g., RNA) to create fragments with cleaved ends at random cleavage locations. Preferably, methods also include reverse transcribing the fragments of RNA in the presence of molecular diversity enhancers (i.e., short random sequences), thereby creating polynucleotides with the molecular diversity enhancers copied therein. The result is a library of polynucleotides that are uniquely identifiable based on combinations of the random cleavage locations and molecular diversity enhancers. Preferably, the method further includes reverse transcription of a fragment of RNA in the presence of a molecular diversity enhancer (i.e., a short random sequence), thereby generating a polynucleotide comprising the copied molecular diversity enhancer. The result is a library of polynucleotides that can be uniquely identified based on the combination of the random cleavage site and the molecular diversity enhancer.
Claims
1. A method for directional tagmentation, comprising: mixing mRNA or total RNA from lysed cells with an oligo; performing a reverse transcription using a reverse transcriptase in the presence of a template switching oligo; synthesizing the first cDNA strand; amplifying the cDNA; subjecting the amplified cDNA to tagmentation and adapter ligation.
2. The method of claim 1, further comprising amplifying the products of the tagmentation.
3. The method of claim 2, wherein the amplifying the products of the tagmentation comprising amplification of 3' ends and sample barcoding.
4. The method of claim 1, wherein the oligo comprises a poly-T sequence.
5. The method of claim 4, wherein the oligo results in libraries enriched for the 3' end of mRNA.
6. The method of claim 1, wherein t the oligo comprises specific primers for amplifying final libraries.
7. The method of claim 6, wherein a specific primer comprises a sequence complementary to an i7 primer.
8. The method of claim 1, wherein the template switching oligo comprises a universal 3' sequence.
9. The method of claim 1, wherein the template switching oligo comprises a molecular diversity enhancer (MDE).
10. The method of claim 9, wherein the MDE comprises a random N-mer.
11. The method of claim 1, wherein the template switching oligo comprises features to prevent concatemerization of the oligo.