Sample to sequence
Inventors
Poritz, Mark Aaron • Ririe, Kirk Max • Pasko, Christopher Paul • Demogines, Ann Michelle • Crisp, Robert John • Rogatcheva, Margarita • Trauscht, Robert Cornelius • Jones, Matthew Kam • Healy, Tyler Lane
Assignees
Biofire Defense LLC • Biofire Diagnostics LLC
Publication Number
US-10718013-B2
Publication Date
2020-07-21
Expiration Date
2036-06-02
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Abstract
Method and sample vessels are provided for amplification and sequencing of nucleic acids in a sample.
Core Innovation
The invention provides methods and sample vessels for amplification and sequencing of nucleic acids in a sample through a two-step process. The method involves placing a sample in an amplification chamber configured to amplify a plurality of individual nucleic acids, subjecting the chamber to amplification conditions, then moving the sample to an array of second-stage amplification wells, each configured for further amplifying a single nucleic acid. A second-stage amplification is performed in these wells to generate amplicons, followed by subjecting the wells to sequencing conditions.
The core problem addressed is the need for rapid, near point-of-care or laboratory-based identification and sequencing of nucleic acids in various applications. Existing single-stage PCR detection systems for complex nucleic acid mixtures often result in multiple non-specific products, requiring additional steps or filters to isolate the correct amplicon. The invention aims to enable rapid sample-to-sequence workflows for situations where swift genetic information is critical, such as pathogen detection, drug resistance identification, organ transplantation, and forensic analysis.
The invention includes optional features such as tethering primers to supports, performing the process within a single container with sealable ports, and using an array of wells or spots for the second-stage amplification. Sequencing can occur directly in these wells without a filter or intermediate identification step, owing to the high specificity and homogeneity achieved by the two-step (nested) amplification process.
Claims Coverage
The patent comprises two independent method claims, each with several inventive features relating to two-step nucleic acid amplification and direct sequencing without intervening filters.
Two-step amplification and sequencing of nucleic acids without filter
A method comprising: - Amplifying nucleic acids in a first mixture using a plurality of first-stage primer pairs, each configured for one target to generate a first-stage amplicon. - Amplifying the first-stage amplicons using a plurality of second-stage primer pairs, each configured for a corresponding first-stage amplicon, with at least one second-stage primer nested within its corresponding first-stage pair, to generate a single molecular species for each target. - Sequencing the single molecular species generated, without using a filter between the second amplification and the sequencing step.
Performing steps in a single container with second-stage amplification wells
All steps of two-step amplification and sequencing are performed in a single container. The first-stage amplification occurs in a designated first-stage chamber. Second-stage amplification and sequencing take place in an array of second-stage amplification wells.
Tethered second-stage primers to a solid support
At least one of each pair of second-stage primers is tethered to a solid support during the second-stage amplification step, which can facilitate downstream sequencing processes.
Option for different reaction vessels for amplification and sequencing
The method allows for second-stage amplification and sequencing to occur in either a single reaction chamber or in different reaction vessels.
Positive call detection for selective sequencing
The method may include detecting whether the first-stage amplicon has been generated in each second-stage amplification well, to generate a positive call for wells where amplification occurs. Sequencing may be performed only on wells with a positive call.
Performing all steps in about 5 hours or less
The entire process—from first-stage amplification to sequencing—can be completed in about 5 hours or less.
No filter is used between first and second amplification
In addition to omitting a filter between the second amplification and sequencing, no filter is used between the first and second amplification steps to identify the correct first-stage amplicon.
The independent claims describe a two-step (nested) amplification and sequencing method that does not require additional filtering between amplification and sequencing, enabling direct sequencing of highly specific amplicons. The method allows for flexible configurations regarding containerization, primer tethering, timing, and the selective application of sequencing based on positive amplification.
Stated Advantages
The two-step amplification yields essentially homogeneous (single molecular species) amplicons, allowing direct sequencing without need for a filter or intermediate identification step.
The method enables rapid sample-to-sequence workflows, with the option for the entire process to be completed in about 5 hours or less.
Methods and systems can be implemented in a closed system to minimize amplicon contamination risk.
The process allows for detection and sequencing from very low copy number targets even in the presence of complex backgrounds of other nucleic acids.
Positive or negative calls based on amplification can be used to earmark which wells or spots undergo sequencing, maximizing efficiency.
Documented Applications
Rapid identification of mutations that confer antibiotic resistance, including characterization of ESBLs in bacterial pathogens.
Sequencing of viral genes for determining virus presence and drug resistance, such as for Hepatitis C, Hepatitis B, HIV, and influenza.
Identification of tissue for forensic purposes, including mapping the length of Short Tandem Repeats (STRs).
Tissue typing for organ transplantation by sequencing MHC genes.
Identification of cytochrome P450 alleles to determine rates of drug metabolism.
Sequencing cancer genes and detecting other infectious agents such as tuberculosis.
Use in clinical diagnostics and other fields such as bioterrorism, food safety, environmental safety, agriculture, and ecology.
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