Targeted whole genome amplification method for identification of pathogens
First Claim
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1. A method comprising:
- a) amplifying at least one pathogen genome from a sample suspected of comprising at least one pathogen genome and at least one background genome using at least one targeted whole genome amplification primer pair, thereby elevating the quantity of nucleic acid representing said at least one pathogen genome relative to the quantity of nucleic acid representing said at least one background genome, wherein said plurality of targeted whole genome amplification primers is selected by;
i) identifying at least one pathogen genome;
ii) identifying at least one background genome;
iii) identifying a plurality of genome sequence segments having unique sequences within said pathogen genome sequence;
iv) determining frequency of occurrence of members of said plurality of genome sequence segments within said pathogen genome sequence and determining frequency of occurrence of said plurality of genome sequence segments within said background genome sequences;
v) calculating a selectivity ratio for said members by dividing said frequency of occurrence within said pathogen genome sequence by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences;
vi) selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value;
vii) determining the lengths of pathogen genome sequence occurring between genome sequence segments of said first sub-set;
viii) selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation distance of less than a selected length of nucleobases; and
ix) selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that, under whole genome amplification conditions, said at least one pathogen genome is amplified selectively over said at least one background genomes;
b) producing one or more amplification products representing bioagent identifying amplicons from said amplified pathogen genome using said at least one primer pair;
c) measuring molecular masses of said amplification products by mass spectrometry;
d) calculating base compositions of said amplification products from said molecular masses without sequencing said amplification products; and
e) comparing said base compositions with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said at least one primer pair, thereby identifying said pathogen in said sample.
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Abstract
The methods disclosed herein relate to methods and compositions for amplifying nucleic acid sequences, more specifically, from nucleic acid sequences of pathogens by targeted whole genome amplification.
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Citations
79 Claims
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1. A method comprising:
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a) amplifying at least one pathogen genome from a sample suspected of comprising at least one pathogen genome and at least one background genome using at least one targeted whole genome amplification primer pair, thereby elevating the quantity of nucleic acid representing said at least one pathogen genome relative to the quantity of nucleic acid representing said at least one background genome, wherein said plurality of targeted whole genome amplification primers is selected by; i) identifying at least one pathogen genome; ii) identifying at least one background genome; iii) identifying a plurality of genome sequence segments having unique sequences within said pathogen genome sequence; iv) determining frequency of occurrence of members of said plurality of genome sequence segments within said pathogen genome sequence and determining frequency of occurrence of said plurality of genome sequence segments within said background genome sequences; v) calculating a selectivity ratio for said members by dividing said frequency of occurrence within said pathogen genome sequence by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences; vi) selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value; vii) determining the lengths of pathogen genome sequence occurring between genome sequence segments of said first sub-set; viii) selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation distance of less than a selected length of nucleobases; and ix) selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that, under whole genome amplification conditions, said at least one pathogen genome is amplified selectively over said at least one background genomes; b) producing one or more amplification products representing bioagent identifying amplicons from said amplified pathogen genome using said at least one primer pair; c) measuring molecular masses of said amplification products by mass spectrometry; d) calculating base compositions of said amplification products from said molecular masses without sequencing said amplification products; and e) comparing said base compositions with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said at least one primer pair, thereby identifying said pathogen in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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36. A method comprising the steps of:
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a) extracting nucleic acids from a sample; and b) mixing said nucleic acids with at least one targeted whole genome amplification primer pair, a high processivity polymerase enzyme to produce an amplification mixture, wherein said plurality of targeted whole genome amplification primers is selected by; i) identifying at least one target genome suspected of being present in said sample; ii) identifying at least one background genome suspected of being present in said sample; iii) identifying a plurality of genome sequence segments having unique sequences within said target genome sequence; iv) determining frequency of occurrence of members of said plurality of genome sequence segments within said target genome sequence and within said background genome sequences; v) calculating a selectivity ratio for said members by dividing said frequency of occurrence within said target genome by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences; vi) selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value; vii) determining the lengths of target genome sequence occurring between genome sequence segments of said first sub-set; viii) selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation of less than a selected length of nucleobases; and ix) selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that said at least one target genome is amplified selectively over said at least one background genome; c) amplifying one or more of said extracted nucleic acids in said mixture of step b wherein said amplifying step is a targeted whole genome amplification reaction; d) performing a second amplification step using at least one second primer pair that defines a bioagent identifying amplicon to obtain at least one second amplification product; e) measuring the molecular mass of said at least one second amplification product by mass spectrometry; f) calculating a base composition of said at least one second amplification product from said molecular mass without sequencing said at least one second amplification product; and g) comparing said base composition with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said at least one second primer pair, thereby identifying said pathogen in said sample. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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Specification