A high throughput sequencing method and kit
AssigneeDiagenode S.A.
InventorsJerome KROONEN, Celine SABATEL, Gilles BROCART, Florence DURIEUX
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Abstract
A high throughput sequencing method and kit The present invention is in the field of diagnostic and sequencing technologies and is related to a high throughput sequencing method and a kit comprising tools for performing this method, that combine a capture and amplification by switching detection step, preferably the so-called “Capture and Amplification by Tailing and Switching” (CATS) and sequencing technology, preferably the so-called “Nanoballs sequencing” technologies.
Claims17
A high throughput sequencing method of a nucleic acid strand sequence comprising the steps of
- providing a sample, single cell sample, comprising a native single stranded nucleic acid sequence or native double stranded nucleic acid sequence,
- possibly fragmenting the native single stranded nucleic acid sequence or native double stranded nucleic acid sequence, into smaller nucleic acid sequence fragments
- possibly denaturing the native double strand nucleic acid sequence(s)
- possibly end-repairing the native nucleic acid sequences,
- hybridizing a priming oligonucleotide sequence complementary to the added nucleotide sequence and synthesizing a cDNA sequence with a template dependent DNA polymerase to obtain a double stranded nucleic acid sequence
- hybridizing a template switching oligonucleotide to the said generated double stranded nucleic acid sequence,
- extending the 3′ end of the cDNA strand to synthesize a double stranded nucleic acid sequence, wherein one strand of the nucleic acid sequence comprises the priming oligonucleotide and a cDNA sequence that is complementary to the single stranded nucleic acid sequence and to the template switching oligonucleotide sequence,
- adding a splint oligo sequence that hybridize to adapter DNA sequences which are ligated to form a circle and adding an exonuclease to remove all remaining single stranded and double stranded DNA products to collect only circular DNA template,
- obtaining DNA nanoballs (DNBs) by performing a rolling circle replication of the synthesized double stranded nucleic acid sequence,
- fixing the DNA nanoballs (DNBs) on a patterned array flow cell,
- performing a base sequencing and
- obtaining an identification, preferably of each nucleotide of the native nucleic acid sequence, by nanopore sequencing or imaging preferably upon a high resolution CCD camera.
A sequencing kit comprising
- a reagent capable of adding nucleotides to the 3-terminus of a single stranded nucleic acid,
- an end-repair enzyme,
- reagents for a genetic amplification, preferably a PCR genetic amplification,
- a reverse transcriptase enzyme,
- a priming oligonucleotide,
- a template switching oligonucleotide and
- a rolling circle replication enzyme
Use of the kit or the method according to anyone of the preceding claims, for sequencing or expression analysis, cloning labelling, for the identification of genes or mutation, in personalized medicine, therapy monitoring, prediction, prognosis, early detection of human or animal disease or forensic science, analysis of infectious diseases and genomes of viruses, bacteria, fungi, animals or plant, including their derived cells, characterization of plants, fruits, breeding checks detection of plants or fruits diseases.
Dependencies are inferred from the claim text.