Synthetic nucleic acid spike-ins
Inventors
Christians, Fred C. • Vilfan, Igor D. • Kertesz, Michael • Blauwkamp, Timothy A. • Venkatasubrahmanyam, Shivkumar • Rosen, Michael • Sit, Rene
Assignees
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Publication Number
US-11692224-B2
Publication Date
2023-07-04
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 nucleic-acid spike-in panels for next-generation sequencing that enable quantification and monitoring of nucleic-acid recovery and absolute abundance. A known quantity of spike-in nucleic acids with sequences unique to each other is added to an initial sample comprising target nucleic acids, and sequencing is performed to obtain target nucleic acid sequence reads and spike-in nucleic acid sequence reads. The spike-in sequence reads are then used to calculate abundance of the target nucleic acids.
The described spike-in designs account for performance dependencies such as fragment length, GC content, and double-stranded DNA library preparation denaturation behavior, including duplex melting temperature (T_m). The disclosure provides a high-granularity GC/length spike-in design with multiple duplexes across different GC values to improve inference of starting quantities of short cfDNA, including pathogen cfDNA, and supports both relative abundance and absolute abundance inference from sequencing library outcomes.
The disclosure introduces high-diversity degenerate spike-in pools to measure loss of sequence diversity as a proxy for abundance loss and to identify processing bottlenecks. It further describes tracer sequences for cross-contamination detection in positive controls and a molecular LIMS approach using spike-in barcoding of reagents, lots, or shipments. Sequencing and bioinformatics context include normalization using spike-ins.
Claims Coverage
The independent claim covers adding a known quantity of uniquely sequenced spike-in nucleic acids, sequencing both target and spike-in portions, and using spike-in sequence reads to calculate target nucleic-acid abundance. The claim set further refines spike-in composition and structure, sample context, sequencing assay scope, and analysis, including host-sequence sequestering or subtraction.
Uniquely sequenced spike-in addition for abundance determination
Determining abundance of nucleic acids in an initial sample by adding a known quantity of spike-in nucleic acids, wherein the spike-in nucleic acids comprise at least 1,000 spike-in nucleic acids with sequences unique to each other.
Sequencing target and uniquely identified spike-in reads
Performing a sequencing assay on a portion of the target nucleic acids and a portion of the at least 1,000 spike-in nucleic acids to obtain target nucleic acid sequence reads and spike-in nucleic acid sequence reads.
Abundance calculation using spike-in sequence reads
Using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids.
High-throughput sequencing assay for determining abundance
Performing the method such that the sequencing assay uses high-throughput sequencing.
Bioinformatic host sequence sequestering or subtraction
Using an initial sample with human sequences and performing bioinformatic analysis by sequestering or subtracting host sequences from the bioinformatic analysis of the sequence reads.
Across the independent claim, abundance is determined by combining uniquely sequenced spike-in nucleic acids with sequencing of both target and spike-in reads, followed by calculating target abundance using spike-in sequence reads. Dependent refinements in the provided claim set specify high-throughput sequencing and host-sequence subtraction in bioinformatic analysis.
Stated Advantages
Enables quantification and monitoring of nucleic-acid recovery and absolute abundance.
Improves inference of starting quantities of short cfDNA, including pathogen cfDNA.
Measures loss of sequence diversity as a proxy for abundance loss to identify processing bottlenecks.
Detects cross-contamination in positive controls using tracer sequences.
Supports normalization using spike-ins.
Enables tracking via a molecular LIMS using spike-in barcoding of reagents, lots, or shipments.
Track samples and reagents and enable cross-contamination detection using identifying tags and tracer sequences.
Improve library preparation yield while evading sequencing by inhibition of ligation, end-repair, or amplification, with optional depletion prior to sequencing.
Documented Applications
Determining abundance of pathogen nucleic acids in host samples using next generation sequencing.
Using spike-ins with NGS assays to determine relative abundance and/or absolute abundance of non-host nucleic acids in host samples during sample processing.
Tracking samples, reagents, reagent lots, and detecting cross-contamination in sequencing workflows using sample identifiers and tracer sequences.
Improving library preparation yield for sequencing assays using modified or engineered carrier spike-ins with sequencing evasion and optional depletion prior to sequencing.
Quantifying and monitoring nucleic-acid recovery and absolute abundance in initial samples using next-generation sequencing spike-in panels.
Inference of starting quantities of short cfDNA, including pathogen cfDNA.
Cross-contamination detection in positive controls using tracer sequences.
Tracking reagents, lots, or shipments using a molecular LIMS based on spike-in barcoding.
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