Multitag sequencing ecogenomics analysis-US
Inventors
Assignees
BIOSPHEREX A Inc IN COMMONWEALTH OF VIRGINIA LLC LLC • Prescient Metabiomics Jv LLC
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Publication Number
US-8603749-B2
Publication Date
2013-12-10
Expiration Date
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.
Core Innovation
The invention provides a multiplex massively parallel sequencing method for determining abundance profiles of one or more target polynucleotide sequences across a plurality of samples. Target polynucleotides are amplified and tagged by PCR in each sample using an amplification primer that includes a high throughput sequencing adaptor, a sample-specific tag sequence of at least four nucleotides in length, and a priming sequence for amplifying the target polynucleotide sequence(s).
The amplified polynucleotides are combined and the polynucleotide pool is sequenced in high throughput to determine the sequence of at least 300 tagged polynucleotides for each sample. The nucleotide sequences are assigned to the originating samples using the nucleotide sequence of the sample-specific tag, thereby determining abundance profiles of the target polynucleotide sequences across the samples.
For microbial community profiling, the invention amplifies and tags target polynucleotides from a plurality of clinical or environmental samples using an amplification primer pair with a high throughput sequencing adaptor, a tag sequence that identifies the sample and is different by at least two nucleotides across the samples, and a priming sequence that is the same in each sample. The priming sequence hybridizes 3′ to a genetic region that is variable across microbial species, followed by high throughput sequencing of the pooled tagged polynucleotides and assignment to originating samples via the tag to determine microbial community profiles across samples.
Claims Coverage
The document includes two independent claims covering (i) a general multiplex method for determining abundance profiles across many samples and (ii) a microbial-community-focused multiplex method for clinical or environmental samples. Each independent claim centers on PCR amplification with sample-specific tagging, high throughput sequencing of a combined tagged pool, and demultiplexing by reading out the sample-identifying tag sequence to determine profiles.
Pcr amplification with high throughput sequencing adaptor and sample-specific tag
Amplifying and tagging target polynucleotides by PCR in each of the 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 the target polynucleotide sequence(s).
High throughput sequencing of combined tagged polynucleotide pool
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 the samples.
Assigning reads to originating samples using sample-specific tag sequences
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.
Microbial community profiling with variable-region priming and sample-identifying tag
Amplifying and tagging target polynucleotides by PCR in each of the plurality of clinical samples or environmental samples with an amplification primer pair comprising a high throughput sequencing adaptor, a tag sequence to identify the sample that 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.
High throughput sequencing and assignment to determine microbial community profiles
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 the samples, and assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the tag thereby determining microbial community profiles across the samples.
Across the independent claims, the coverage focuses on PCR amplification with high throughput sequencing adaptors and sample-identifying tags, high throughput sequencing of a combined tagged pool to obtain at least 300 tagged polynucleotides per sample, and assigning nucleotide sequences back to originating samples via the tag sequence to produce abundance profiles or microbial community profiles.
Stated Advantages
Enables determining abundance profiles of target polynucleotide sequences across many samples using tag assignment from sequenced reads.
Enables determining microbial community profiles across clinical samples or environmental samples using variable-region priming and tag-based assignment.
Documented Applications
Microbial community profiling across a plurality of clinical samples or a plurality of environmental samples.
Quantifying abundance profiles of one or more target polynucleotide sequences across a plurality of samples.
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