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EP-4540411-B1 - METHOD FOR THE TARGETED INTEGRATION OF UNIQUE MOLECULAR IDENTIFIERS (UMIS) AND THE HIGH PRECISION DETECTION OF MEDICAL CONDITIONS

EP4540411B1EP 4540411 B1EP4540411 B1EP 4540411B1EP-4540411-B1

Inventors

  • CARRASCO, JAVIER

Dates

Publication Date
20260513
Application Date
20240124

Claims (12)

  1. A high precision method for the detection of a medical condition, comprising: A. Providing a targeted DNA sequence (10) that has been obtained from patient sample comprising the targeted DNA sequence (10); wherein the targeted DNA sequence is from 20 to 200 bases upstream and from 20 to 200 base pairs downstream from the center of a region of interest (ROI, 20), wherein the ROI (20) consists of 1 to 100, preferably from 10 to 50, and even more preferably from 20 to 50 bases; and wherein the ROI (20) is associated with the medical condition; and attaching a unique molecular identifier (UMI, 33, 33') to the targeted DNA sequence (10) using a UMI integration primer (30, 30'); wherein the UMI integration primer (30, 30') comprises: ▪ an amplification adapter (31, 31'), ▪ a Unique Molecular Identifier (UMI, 33, 33'), ▪ a sample identifier (32, 32') ; and ▪ a DNA sequence specific for the targeted DNA sequence (34, 34'), wherein 3'-end is neighboring the targeted DNA sequence (10). B. Amplifying the targeted DNA sequence (10) through a first enrichment PCR thereby obtaining a first enrichment amplicon of 200 to 700 base pairs, wherein the first enrichment amplicon comprises the ROI (20); C. Amplifying the targeted DNA sequence (10) through a second nested enrichment PCR thereby obtaining a second enrichment amplicon of 200 to 500 base pairs, wherein the second enrichment amplicon comprises the ROI (20); D. Attaching NGS adapters (60, 61) to the second enrichment PCR amplicons; and E. Sequencing the second enrichment amplicons through pair-end sequencing as Next Generation Sequencing (NGS); wherein the first enrichment PCR comprises amplification with: ▪ one or more first enrichment PCR primers (40), wherein the first enrichment PCR primers (40) are specific for the targeted DNA sequence (10); wherein the first enrichment PCR primers (40) are forward if the UMI integration primer (30) is downstream from the ROI (20); and wherein the first enrichment PCR primers are reverse if the UMI integration primer (30') is upstream from the ROI (20); and ▪ a primer (41) complementary to the amplification adapter (31); wherein the second enrichment PCR comprises amplification with: ▪ one or more second enrichment PCR primers (50), wherein the second enrichment PCR primers (50) are specific for the ROI (20) or for the targeted DNA sequences (10), wherein the second enrichment PCR primers (50) are nested with respect to first enrichment PCR primers (40); and wherein the second enrichment PCR primer (50) comprises a generic adapter (51) at the 5'-end; and ▪ a primer (52) complementary to the amplification adapter (31), wherein the primer (52) comprises a generic adapter (53).
  2. The method of claim 1, wherein the targeted DNA sequence (10) has a length of 20 to 200 nucleotides, preferably 20 to 100 nucleotides from the center of the ROI.
  3. The method of any one of the preceding claims, wherein the ROI (20) consists of 1 to 100, preferably from 10 to 50, and even more preferably from 20 to 50 bases.
  4. The method of any one of the preceding claims, wherein the DNA sequence specific for the targeted DNA sequence (34, 34') has a length of 10 to 60 nucleotides, preferably 15 to 25 nucleotides, and even more preferably 18 to 24 nucleotides.
  5. The method of any one of the preceding claims, wherein the UMI has a length of 5 to 20 nucleotides, preferably 8 to 18 nucleotides, even more preferably 12 to 15, and even more preferably 14 nucleotides.
  6. The method of any one of the preceding claims, wherein the UMI is an aleatory sequence, a semi-aleatory sequence or a combination thereof.
  7. The method of any one of the preceding claims, wherein the random portion of the semi-aleatory sequence is from 2 to 12 bases.
  8. The method of any one of the preceding claims, wherein the UMI consists of the following consecutive base order: ▪ 4 random bases, ▪ one fixed base, ▪ 4 random bases, ▪ one fixed base, and ▪ 4 random bases.
  9. The method of any one of the preceding claims, wherein the region of interest is a T-cell receptor or a B-cell receptor, preferably a T-cell receptor.
  10. The method of any one of the preceding claims, wherein the medical condition is cancer, preferably breast cancer or gastrointestinal cancer.
  11. The method of any one of the preceding claims, wherein medical condition is recidivist cancer.
  12. The method of any one of the preceding claims further comprising the determination of a treatment in function of the detected region of interest. ▪

Description

FIELD OF THE INVENTION The present invention concerns the field of high precision detection of medical or cosmetic conditions, in particular the detection of cancer. BACKGROUND Cancer is a complex multigenic disease. Diverse genetic alterations characterize this condition. High precision diagnostics help detect the onset, progression or recidivity of cancer. This facilitates a more personalized approach to clinical care through risk stratification and treatment selection. Next-generation sequencing (NGS) allows for the determination of unique gene expression signatures for each tumor. This signature contributes to both disease classification, diagnostics, and prognostics. For example, US2022195493A1 to Cellecta describes the multiplex targeted PCR through the preparation of barcoded gene specific DNA fragments. The method includes employing a set of gene specific primer pairs, wherein each pair of gene specific primers is made up of a forward primer and a reverse primer, at least one of which includes a sample barcode domain. The sample barcoded nucleic acid has the following structure: 3-linker 1-sample barcode domain-anchor 2 domain-RNA binding domain-5. However, this document does not describe a method comprising the integration of UMI integration primer, followed by two consecutive nested enrichment PCR amplifications. US2021355533A1 to Tempus Labs discloses profiling T cell receptor (TCR) and B cell receptor (BCR) repertoire using next-generation sequencing (NGS) methods and hybrid-capture probes. The hybrid capture probes comprise, in order from 5' to 3', an amplification region, a sample tag region, a UMI, and an anchor region. Therefore, there is still a need for further improved cancer diagnostics. US20210139970A1 discloses the integration of a UMI. But this UMI is far removed from the region of interest, see Figure 3. US20190078148A1 describes the addition of a UMI in a non-target approach, [0012]. US20210355533A1 describes the integration of a UMI with a "non-targeted" approach, [0144]. SHORT DESCRIPTION OF THE INVENTION The inventors have surprisingly found that the precision to detect and characterize T cell receptors (TCR) diversity is substantially increased by ▪ integrating the UMI upstream or downstream of a region of interest (ROI) and▪ performing a NGS library enrichment using 2 nested ROI specific PCR amplifications. This is particularly relevant for TCR monitoring applications such as cancer. Accordingly, a first aspect of the invention is A high precision method for the detection of a medical condition, comprising: A. Providing a targeted DNA sequence (10) that has been obtained a patient sample comprising a targeted DNA sequence (10); wherein the targeted DNA sequence is from 20 to 200 bases upstream and from 20 to 200 base pairs downstream from the center of a region of interest (ROI, 20), wherein the ROI (20) consists of 1 to 100, preferably from 10 to 50, and even more preferably from 20 to 50 bases; and wherein the ROI (20) is associated with the medical condition and attaching a unique molecular identifier (UMI, 33, 33') to the targeted DNA sequence (10) using a UMI integration primer (30, 30'); wherein the UMI integration primer (30, 30') comprises: ▪ an amplification adapter (31, 31'),▪ a Unique Molecular Identifier (UMI, 33, 33'),▪ a sample identifier (32, 32') ; and▪ a DNA sequence specific for the targeted DNA sequence (34, 34'), wherein 3'-end is neighboring the targeted DNA sequence (10).E. Sequencing the second enrichment amplicons through pair-end sequencing as Next Generation Sequencing (NGS); wherein the first enrichment PCR comprises amplification with: ▪ one or more first enrichment PCR primers (40), wherein the first enrichment PCR primers (40) are specific for the targeted DNA sequence (10); wherein the first enrichment PCR primers (40) are forward if the UMI integration primer (30) is downstream from the ROI (20); and wherein the first enrichment PCR primers are reverse if the UMI integration primer (30') is upstream from the ROI (20); and▪ a primer (41) complementary to the amplification adapter (31);wherein the second enrichment PCR comprises amplification with: ▪ one or more second enrichment PCR primers (50), wherein the second enrichment PCR primers (50) are specific for the ROI (20) or for the targeted DNA sequences (10), wherein the second enrichment PCR primers (50) are nested with respect to first enrichment PCR primers (40); and wherein the second enrichment PCR primer (50) comprises a generic adapter (51) at the 5'-end; and▪ a primer (52) complementary to the amplification adapter (31), wherein the primer (52) comprises a generic adapter (53). In another aspect, the targeted DNA sequence (10) has a length of 20 to 200 nucleotides, preferably 20 to 100 nucleotides from the center of the ROI. In another aspect, the ROI (20) consists of 1 to 100, preferably from 10 to 50, and even more preferably from 20 to 50 bases. In another aspect, the DNA sequence spe