Synthetic Nucleic Acid Spike-Ins for Low-Abundance Sequencing Quantification
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Solution Overview
Problem
Next generation sequencing methods face inefficiencies and inaccuracies in detecting and quantifying low abundance nucleic acids, particularly in complex clinical samples.
Innovation Solution
The use of spike-in synthetic nucleic acids with unique sequences, lengths, and GC content to improve detection and quantification by calculating diversity loss and comparing it with the starting quantity to determine target nucleic acid abundance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If next generation sequencing is used to analyze complex clinical samples, then massive amounts of genetic data can be gathered, but detection and quantification of low abundance nucleic acids becomes inefficient and inaccurate
Solution Approach 1:
The patent introduces synthetic spike-in nucleic acids as intermediary reference materials that are added to clinical samples before processing. These synthetic nucleic acids serve as mediators to track and correct for losses during sample processing, enabling accurate quantification of low abundance pathogen nucleic acids by comparing their recovery against the known input amounts of spike-ins
Solution Approach 2:
The patent employs spike-in nucleic acids with deliberately varied parameters including different sequences, lengths, and GC contents. By changing these parameters across multiple spike-in variants, the method captures different biases introduced during sample processing, allowing for more comprehensive correction and improved detection accuracy of target nucleic acids
2Device complexity
If conventional sequencing methods are used without spike-ins, then the process is simpler, but quantification of target nucleic acids is inaccurate due to uncorrected processing losses
Solution Approach 1:
The patent applies preliminary action by adding spike-in nucleic acids to the sample at the very beginning of the processing workflow, before any extraction, amplification, or sequencing steps. This early introduction allows the spike-ins to experience the same processing conditions as the target nucleic acids, enabling accurate tracking and correction of losses throughout the entire process
3Measurement precision
If more synthetic spike-in nucleic acids with diverse features are added to correct for processing losses, then quantification accuracy improves, but the complexity of the assay increases
Solution Approach 1:
The patent designs spike-in nucleic acids with universal features that serve multiple functions simultaneously: they contain unique sequences for identification, varied lengths for capturing size-related biases, different GC contents for correcting composition-related losses, and known concentrations for quantification. This multi-functionality allows a single spike-in mixture to address multiple sources of bias without requiring separate control experiments
Data Source
AI summary
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.