METHOD FOR PRODUCING A POPULATION OF SYMMETRICALLY BARCODED TRANSPOSOMES
Researchers at UCSF have developed a method for producing a population of symmetrically barcoded transposomes that allow the reconstruction of a tagmented nucleic acid sequence without relying on alignment to a reference sequence.
Many next generation sequencing workflows rely on “tagmenting” a sample sequence, whereby said sequence is cleaved by a transposase and double stranded adaptors containing transposon end sequence are added to each of the cleaved ends. The fragments can then be amplified with PCR and subsequently sequenced. Many iterations of this method have been developed and expanded upon, however the issue with these methods is that the information about which fragments were next to each other in the unfragmented nucleic acid is lost after tagmentation. This drawback makes it impossible to reconstruct the unfragmented sequences without relying on alignment to a reference sequence.
Stage of Research
The inventors have developed a method for producing a population of symmetrically barcoded transposome complexes that are loaded with a pair of identical double stranded adapters. During tagmentation, each transposome complex should make a double-stranded break in the substrate and add the same barcode to the newly created nucleic acid fragment ends. This in turn facilitates assembly of the sequences, because adjacent fragments have the same barcoded adapters on their ends.
Applications
- Analyzing genomic DNA, cDNA/RNA hybrids, and double-stranded cDNA from virtually any organism.
Advantages
- This method combines all the benefits of previous tagmentation methods (including ease of use, relative unbiased cleaving of template nucleic acid sequences, barcoding, and easy amplification of the cleaved sequence) with the ability to reconstruct the cleaved sequence based solely on the barcoded adaptors and without the need for a reference sequence.
- New and unknown sequences and genomes can be more easily sequenced and reconstructed.
Stage of Development
Research – in vitro
Publications
WO2022/256223
Related Web Links
N/A
Keywords
Tagmentation, transposase, adaptor, barcoding, alignment, sequence reconstruction, Next-Generation Sequencing
Technology Reference
CZ Biohub ref. no. CZB-209F; UCSF ref. no. SF2021-223
