Synthetic nucleic acid spike-ins
Inventors
Christians, Fred C. • Vilfan, Igor D. • Kertesz, Michael • Blauwkamp, Timothy A. • Venkatasubrahmanyam, Shivkumar • Rosen, Michael • Sit, Rene
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9976181-B2
Publication Date
2018-05-22
Expiration Date
Abstract
This disclosure provides methods for determining relative abundance of one or more non-host species in a sample from a host. Also provided are methods involving addition of known concentrations of synthetic nucleic acids to a sample and performing sequencing assays to identify non-host species such as pathogens. Also provided are methods of tracking samples, tracking reagents, and tracking diversity loss in sequencing assays.
Core Innovation
The invention provides a method for determining abundance of nucleic acids in an initial sample comprising target nucleic acids. The method adds a starting quantity of at least 1,000 unique synthetic nucleic acids, where each synthetic nucleic acid comprises an identifying tag and a variable region comprising at least 5 degenerate bases. The synthetic nucleic acids function as tracking molecules within a sequencing assay workflow.
The method performs a sequencing assay on a portion of the target nucleic acids and on a portion of the at least 1,000 unique synthetic nucleic acids, obtaining target and synthetic nucleic acid sequence reads. A diversity loss value is calculated for the portion of the at least 1,000 unique synthetic nucleic acids. The abundance of the target nucleic acids in the initial sample is determined using the diversity loss value.
The approach addresses quantification and QC using pools of unique synthetic nucleic acids and identifying tags to monitor diversity loss and compute abundance as absolute abundance and relative abundance, including for pathogen nucleic acids. Synthetic constructs that include tags and degeneracy are used to monitor bottlenecks and sample processing loss.
Claims Coverage
The partial content includes one independent claim. Three inventive features are extracted from the claim set.
Unique synthetic nucleic-acid spike pool with identifying tags and degenerate variable regions
Adding a starting quantity of at least 1,000 unique synthetic nucleic acids to the initial sample, wherein each synthetic nucleic acid comprises an identifying tag and a variable region comprising at least 5 degenerate bases.
Sequencing both target nucleic acids and the synthetic nucleic acids to obtain reads
Performing a sequencing assay on a portion of the target nucleic acids and on a portion of the at least 1,000 unique synthetic nucleic acids, thereby obtaining target and synthetic nucleic acid sequence reads.
Diversity-loss-based determination of target nucleic-acid abundance
Calculating a diversity loss value for the portion of the at least 1,000 unique synthetic nucleic acids, wherein abundance of the target nucleic acids in the initial sample is determined using the diversity loss value.
The inventive features are the unique synthetic nucleic-acid spike pool, sequencing of both targets and spikes to generate reads, and determination of target abundance using diversity loss from the synthetic nucleic acids.
Stated Advantages
Enables inference of denaturation-driven recovery losses in sequencing sample QC and quantification.
Enables absolute abundance estimation of target nucleic acids using a diversity loss value derived from synthetic nucleic-acid reads.
Enables determination of diversity loss to identify library bottlenecks and detect duplicate recovery.
Monitors cross-contamination using tracer sequences that do not hybridize to sample.
Documented Applications
NGS sample QC and quantification using nucleic-acid spike-in and tracer systems.
Absolute abundance determination for nucleic acids in an initial sample comprising target nucleic acids.
Sequencing-library QC and diagnosis of library bottlenecks and duplicate recovery via degenerate diverse synthetic spike-ins.
Cross-contamination detection in pathogen testing by using tracer sequences as positive control pathogen stocks.
Pathogen genomic DNA control context described as part of the tracer sequence approach for monitoring cross-contamination.
Interested in licensing this patent?