AUTOMATED SAMPLE TO NGS LIBRARY PREPARATION
The present invention generally-relates to cartridges for automated systems and automated methods for fully automated processing of biological samples to next-generation sequencing-ready nucleic acid libraries. In particular, the present invention concerns a fluidic cartridge comprising a compartment for receiving a biological sample, means for liberating and/or purifying nucleic acids from the received sample and for transporting said nucleic acids to a compartment wherein NGS library can be prepared from said nucleic acids using provided therein reagents. In a particular aspect, the present invention also concerns cartridges for automated systems and automated methods for preparing an NGS nucleic acid library from a biological sample simultaneously or sequentially with a qPCR assay performed on the same biological sample.
1 . A cartridge for automated processing a biological sample, the cartridge comprising:
a) a sample compartment for receiving a biological sample;
b) means for liberating or purifying nucleic acid from the biological sample received in the sample compartment, said means capable of entering in fluid communication with said sample compartment; and
c) a library compartment for preparing a nucleic acid library positioned downstream of the sample compartment and of the means for liberating or purifying nucleic acid, the library compartment configured to receive at least a portion of the liberated or purified nucleic acid and comprising reagents for preparing a nucleic acid library.
2 . A cartridge according to claim 1 , said cartridge further comprising at least one thermocycling qPCR compartment positioned downstream of the sample compartment and of the means for liberating or purifying nucleic acid, and configured to receive at least a portion of the liberated or purified nucleic acid or at least a portion of the nucleic acid library prepared in the library compartment, wherein said thermocycling qPCR compartment is suitable for amplifying nucleic acids and for allowing detection of signals generated during such amplification and comprises reagents for performing qPCR.
3 . A cartridge according to claim 2 , wherein the reagents for performing qPCR comprise at least one set of primers, preferably a plurality of sets of primers for amplifying at least one disease-associated target nucleic acid sequence.
4 . A cartridge according to claim 1 , said cartridge further comprising at least a second one thermocycling qPCR compartment positioned downstream of the sample compartment and of the means for liberating or purifying nucleic acid, the at least second one thermocycling qPCR compartment configured to receive at least a portion of the liberated or purified nucleic acid or a portion of the nucleic acid library prepared in the library compartment, and comprising a at least one set of primers for performing a nucleic acid quality control (QC) qPCR.
5 . A cartridge according to claim 4 , comprising a plurality of sets of primers for generating amplicons of different sizes.
6 . A cartridge according to claim 1 , wherein the reagents for preparing a nucleic acid library allow:
i) generation of nucleic acid fragments from the portion of the liberated or purified nucleic acid received into the library compartment, and
ii) attaching oligonucleotide adapters to at least one, preferably both ends of said generated nucleic acid fragments.
7 . A cartridge according to claim 6 , wherein the generation of nucleic acid fragments is performed by a PCR, further referred to as “library PCR”, and wherein the attaching oligonucleotide adapters to the library-PCR-generated nucleic acid fragments is performed by including an adapter sequence in a sequence of at least one primer used in said library PCR.
8 . A cartridge according to claim 1 , further comprising a cartridge-specific identifier for storing cartridge-specific information.
9 . A cartridge according to claim 1 , further comprising a recovery compartment positioned downstream of the sample compartment, said recovery compartment providing for recovering any of the following:
i) a portion of the biological sample received into the sample compartment;
ii) a portion of the nucleic acid liberated or purified from the biological sample received into the sample compartment;
iii) at least a portion of the nucleic acid library prepared in the library compartment.
10 . An automated method for preparing a next-generation sequencing (NGS) nucleic acid library from a biological sample, said method comprising the steps of:
a) receiving a biological sample;
b) liberating or purifying at least a part of nucleic acids from the received biological sample;
c) providing at least a portion of the liberated or purified nucleic acid into a library compartment for preparing an NGS nucleic acid library and comprising reagents for preparing a nucleic acid library; and
d) preparing an NGS nucleic acid library;
wherein at least the steps b) to d) are performed on an automated system.
11 . An automated method according to claim 10 , the method further comprising the step of performing qPCR on said automated system either sequentially or simultaneously with respect to the step of preparing the NGS nucleic acid library, wherein said qPCR is performed on a second portion of the liberated or purified nucleic in a thermocycling qPCR compartment suitable for amplifying nucleic acids and allowing detection of signals generated during such amplification.
12 . An automated method according to claim 11 , wherein said at least one qPCR amplifies at least one disease-associated target nucleic acid sequence, or is a quality control (QC) qPCR.
13 . An automated method according to claim 10 , the method performed on a cartridge engageable with the automated system.
14 . An automated method according to claim 13 , wherein the cartridge comprises a cartridge-specific identifier for storing cartridge-specific information and wherein the method further comprises the step of introducing to the automated system the information stored in the cartridge-specific identifier.
15 . Use of a cartridge according to claim 1 for automated preparation of a next-generation sequencing (NGS) library from a biological sample.