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US 11,827,929 B2Granted
Optimized clinical sample sequencing
AssigneeBiocartis NV
InventorsGeert Meersseman, Nicolas Vergauwe
Life of this patent
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Abstract
The present invention generally concerns an automated system capable of performing quantitative PCR (qPCR) analysis of a nucleic acid present in a biological sample together with preparation of a sequencing-ready nucleic acid library from the sample, either simultaneously or sequentially. In a further aspect, the present invention also provides a method for performing qPCR of a nucleic acid present in a biological sample together with simultaneous of sequential preparation of a sequencing-ready nucleic acid library from the sample. Finally, the present invention also provides removable cartridges for use in the automated systems and methods according to the invention.
Claims8
An automated system for quantitative PCR (qPCR) analysis of a nucleic acid present in a biological sample received into said system and for concomitant preparation of a sequencing nucleic acid library from said biological sample, the system comprising:
- a cartridge;
- the cartridge comprising:
- a) at least one sample-receiving compartment for receiving the biological sample, the sample-receiving compartment comprising a silica membrane and at least one additional reagent for liberating or purifying nucleic acid from the received biological sample;
- b) a qPCR compartment in fluid connection with the sample-receiving compartment, the qPCR compartment suitable for amplifying nucleic acids and having a wall transparent to light allowing detection of signals generated during such amplification, said qPCR compartment comprising reagents for performing a multiplex quality control (QC) qPCR adapted to generate data suitable for assessing the quality of nucleic acid subjected thereto,
- wherein the reagents for performing the multiplex QC qPCR comprise at least two primer pairs designed for generating at least two amplicons of discernably different sizes,
- wherein the first one of the at least two primer pairs is adapted to generate an amplicon within the range from 50 to 110 bp, and wherein the second one of the at least two primer pairs is adapted to generate an amplicon within the range from 300 to 550 bp;
- c) a library compartment in fluid connection with the sample-receiving compartment, the library compartment comprising reagents for preparing a sequencing nucleic acid library wherein said reagents for preparing a sequencing nucleic acid library comprise oligonucleotide adapters;
- wherein the sample-receiving compartment, the qPCR compartment, and the library compartment are each comprised as separate compartments in the cartridge;
- and
- d) a mechanical arrangement for dividing the nucleic acid liberated or purified from said biological sample in the sample-receiving compartment between at least the qPCR compartment and the library compartment, wherein the qPCR compartment and the library compartment are both downstream of the sample-receiving compartment and are not in fluid connection with each other;
- wherein the system is configured to run the multiplex QC qPCR and the nucleic acid library preparation simultaneously by individually and in parallel controlling thermocycling conditions at the qPCR compartment and at the library compartment, such that different thermocycling conditions may be used in the qPCR compartment and the library compartment;
- and wherein the system is adapted to assess nucleic acid quality from the data obtained from said multiplex QC qPCR wherein the quality assessment comprises assessment of the degree of fragmentation of the nucleic acid.
9 more elements
An automated system for quantitative PCR (qPCR) analysis of a nucleic acid present in a biological sample received into said system and for concomitant preparation of a sequencing nucleic acid library from said biological sample, the system comprising:
- a cartridge;
- the cartridge comprising:
- a) at least one sample-receiving compartment for receiving the biological sample, the sample-receiving compartment comprising a silica membrane and at least one additional reagent for liberating or purifying nucleic acid from the received biological sample;
- b) a qPCR compartment in fluid connection with the sample-receiving compartment, the qPCR compartment suitable for amplifying nucleic acids and having a wall transparent to light allowing detection of signals generated during such amplification, said qPCR compartment comprising reagents for performing a first multiplex quality control (QC) qPCR adapted to generate data suitable for assessing the quality of nucleic acid subjected thereto,
- wherein the reagents for performing the multiplex QC qPCR comprise at least two primer pairs designed for generating at least two amplicons of discernably different sizes,
- wherein the first one of the at least two primer pairs is adapted to generate an amplicon within the range from 50 to 110 bp, and wherein the second one of the at least two primer pairs is adapted to generate an amplicon within the range from 300 to 550 bp;
- and
- c) a library compartment in fluid connection with the sample-receiving compartment, the library compartment comprising reagents for preparing a sequencing nucleic acid library wherein said reagents for preparing a sequencing nucleic acid library comprise oligonucleotide adapters;
- and
- wherein the sample-receiving compartment, the qPCR compartment, and the library compartment are each comprised as separate compartments in the cartridge;
- d) a mechanical arrangement for dividing the nucleic acid liberated or purified from said biological sample in the sample-receiving compartment between at least the qPCR compartment and the library compartment, wherein the qPCR compartment and the library compartment are both downstream of the sample-receiving compartment;
- and
- e) a fluid channel configured to transfer at least a part of the sequencing nucleic acid library prepared from the library compartment to the qPCR compartment;
- wherein the system is configured to run the first multiplex QC qPCR and the nucleic acid library preparation simultaneously by individually and in parallel controlling thermocycling conditions at the qPCR compartment and at the library compartment, such that different thermocycling conditions may be used in the qPCR compartment and the library compartment;
- wherein the system is configured to run a second multiplex QC qPCR on the sequencing nucleic acid library;
- and wherein the system is adapted to assess nucleic acid quality from the data obtained from said first and second multiplex QC qPCRs wherein the quality assessment comprises assessment of the degree of fragmentation of the nucleic acid.
13 more elements
Dependencies are inferred from the claim text.