Sample series to differentiate target nucleic acids from contaminant nucleic acids
Inventors
Blauwkamp, Timothy A. • HUIJSE, Liza • Rosen, Michael • Vilfan, Igor D.
Assignees
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Publication Number
US-11674167-B2
Publication Date
2023-06-13
Expiration Date
Abstract
This disclosure provides methods, compositions and kits for determining if nucleic acids detected in a sample such as a clinical sample are derived from contaminant pathogens or clinically-relevant pathogens.
Core Innovation
The invention provides a method of processing a sample containing target cell-free nucleic acids by using a dilution series. The method includes a first diluted sample and at least a second diluted sample, where the second diluted sample has a lower concentration of the target cell-free nucleic acids compared to the first diluted sample, or comprises no target cell-free nucleic acids. Sample processing is performed on both diluted samples because contaminant nucleic acids are possibly introduced during the sample processing.
The method quantifies cell-free nucleic acids within the first and second diluted samples by performing a sequencing assay on portions of the cell-free nucleic acids to produce a first quantitative value and a second quantitative value, if present. The method then compares the first quantitative value with the second quantitative value. A decrease in the second quantitative value compared to the first quantitative value confirms a presence of the target cell-free nucleic acids in the sample.
In related embodiments, the method is implemented using a sample series comprising a plurality of samples with different concentrations or quantities of target nucleic acids. Sample processing is performed across the at least two samples, where contaminant nucleic acids and target nucleic acids are derived from the same type of bacteria, fungi, viruses, or protozoa. Quantifying by sequencing and comparing relative quantities distinguishes the target nucleic acids from possible contaminant nucleic acids.
Claims Coverage
The document includes two independent claims that provide dilution-series and sample-series workflows for distinguishing target cell-free nucleic acids from contaminant nucleic acids using sequencing assay quantification and quantitative comparisons. Across the independent claims, the document centers on comparing sequencing-derived quantitative values or relative quantities across differently concentrated sample members to confirm target nucleic acids in the presence of possible contaminant nucleic acids.
Dilution series confirmation of target cell-free nucleic acids
Providing a dilution series with a first diluted sample containing a quantity of target cell-free nucleic acids and a second diluted sample having a lower concentration or comprising no target cell-free nucleic acids; performing sample processing on both diluted samples where contaminant nucleic acids are possibly introduced; quantifying cell-free nucleic acids in the first and second diluted samples by sequencing assay to produce first and second quantitative values; comparing the first quantitative value with the second quantitative value such that a decrease in the second quantitative value compared to the first quantitative value confirms a presence of the target cell-free nucleic acids in the sample.
Sample-series relative quantity distinguishing with same-source contaminant
Providing a sample series comprising a plurality of samples where at least two samples may comprise different concentrations or quantities of target nucleic acids; performing sample processing on at least two of the plurality of samples where contaminant nucleic acids are possibly introduced and contaminant and target nucleic acids are derived from the same type of bacteria, fungi, viruses, or protozoa; quantifying nucleic acids within at least two samples by performing a sequencing assay; comparing relative quantities of the nucleic acids in the at least two samples to distinguish the target nucleic acids from the possible contaminant nucleic acids.
Across the independent claims, the inventive coverage centers on using sequencing-derived quantitative comparisons across dilution series or sample series to distinguish target nucleic acids from possible contaminant nucleic acids.
Stated Advantages
A decrease in the second quantitative value compared to the first quantitative value confirms a presence of the target cell-free nucleic acids in the sample.
Relative quantities of nucleic acids in the at least two samples distinguish target nucleic acids from possible contaminant nucleic acids.
Documented Applications
Distinguishing target cell-free nucleic acids including pathogen nucleic acids from contaminant nucleic acids in a dilution series by comparing sequencing quantitative values across dilutions.
Distinguishing target nucleic acids from possible contaminant nucleic acids in a sample series where contaminant nucleic acids and target nucleic acids are derived from the same type of bacteria, fungi, viruses, or protozoa.
Organism identification by aligning sequence reads to reference genomes for inferred origin of microbes.
Use of control sample options during sample processing of the first and second diluted samples, including plasma from an asymptomatic human subject, a synthetic sample mimicking human plasma, or a sterile sample.
Excluding sequencing of a common sequence of the 16S rRNA gene.
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