Multitag sequencing ecogenomics analysis-US
AssigneeBiospherex, LLC
InventorPatrick M. Gillevet
Life of this patent
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Abstract
Embodiments of the invention herein described relate to multiplex polynucleotide sequence analysis without the use of size separation methods or blotting. In certain particulars the invention relates to multiplex sequencing using massively parallel sequencing methods, such as pyrosequencing methods and sequencing by synthesis. The invention provides increased throughput, increased accuracy of enumerating sample components, and the ability to analyze greater numbers of samples simultaneously or serially on presently available systems, as well as others yet to be developed. In certain of its embodiments the invention relates to the analysis of complex microbial communities, particularly to in-depth analysis thereof in large numbers of samples.
Claims26
A multiplex method for determining abundance profiles of one or more target polynucleotide sequences across a plurality of samples, comprising:
- amplifying and tagging target polynucleotides by PCR in each of said plurality of samples with an amplification primer comprising a high throughput sequencing adaptor, a sample-specific tag sequence of at least four nucleotides in length, and a priming sequence to amplify said target polynucleotide sequence(s);
- combining the amplified polynucleotides and sequencing the polynucleotide pool in high throughput, so as to determine the sequence of at least 300 tagged polynucleotides for each of said samples;
- assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the sample-specific tag, thereby determining abundance profiles of the target polynucleotide sequence(s) across the samples.
A method for determining microbial community profiles across a plurality of clinical samples or a plurality of environmental samples, comprising:
- amplifying and tagging target polynucleotides by PCR in each of said plurality of samples with an amplification primer pair comprising:
- a high throughput sequencing adaptor,
- a tag sequence to identify the sample, and which is different by at least two nucleotides across the samples, and
- a priming sequence that is the same in each sample, the priming sequence hybridizing 3′ to a genetic region that is variable across microbial species;
- combining the amplified polynucleotides and sequencing the polynucleotide pool in high throughput, so as to determine the sequence of at least 300 tagged polynucleotides for each of said samples;
- assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the tag, thereby determining microbial community profiles across the samples.
Dependencies are inferred from the claim text.