Multitag sequencing ecogenomics analysis
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Publication Number
US-11091813-B2
Publication Date
2021-08-17
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 disclosed invention provides multitag multiplex sequencing for determining microbial community profiles across at least five biological samples. For each sample, a 16S rRNA or 18S rRNA variable region is amplified by PCR or linear amplification and is tagged to include a sequencing adaptor, a sample-identifying tag sequence, and a probe sequence. The tag sequences identify which sample a read originates from.
Amplified polynucleotides are combined into a pool and sequenced using a sequencing-by-synthesis sequencer. Nucleotide sequences or subsequences thereof are then assigned to the originating samples based on the nucleotide sequence of the tags. The assigned sequences are aligned to microbial rRNA reference sequences in order to determine microbial community profiles across the samples.
The invention further emphasizes assigning sequences through tag identification together with alignment to microbial rRNA reference sequences, including use of microbial rRNA variable-region matching to reference databases. It supports microbial community profiling without gel electrophoresis, Southern blot, or size separation, and it includes quantification of variant abundances via normalized counts.
Claims Coverage
The provided document includes two independent method claims, one PCR-based and one linear-amplification-based. Across both independents, the claims share five main inventive elements: amplification of an rRNA variable region with a common probe and sample-identifying tags, pooling, sequencing-by-synthesis, assigning reads to samples by tag sequences, and aligning to microbial rRNA reference sequences to determine microbial community profiles across at least five biological samples.
Tagging rRNA variable regions with sample-identifying adaptor-tag-probe constructs
Amplifying and tagging a 16S rRNA or 18S rRNA variable region by PCR for each sample using an amplification primer pair comprising a sequencing adaptor, a tag sequence to identify the sample, and a probe sequence that is the same for each primer pair.
Pooling tagged amplicons and sequencing by synthesis
Combining the amplified polynucleotides and sequencing the polynucleotide pool using a sequencing-by-synthesis sequencer.
Assigning reads to originating samples by tag sequences and aligning to microbial rRNA references
Assigning the nucleotide sequences to originating samples by the nucleotide sequence of the tags and aligning the nucleotide sequences or subsequences thereof to microbial rRNA reference sequences, thereby determining microbial community profiles across the samples.
Linear amplification and tagging of an rRNA variable region with a common probe and sample-identifying tags
Amplifying and tagging a 16S rRNA or 18S rRNA variable region by linear amplification for each sample with an amplification primer comprising a sequencing adaptor, a tag sequence to identify the sample, and a probe sequence that is the same for each primer.
Assigning reads to samples and determining community profiles using tag-based demultiplexing and rRNA reference alignment
Assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the tags and aligning the nucleotide sequences or subsequences thereof to microbial rRNA reference sequences, thereby determining microbial community profiles across the samples.
Both independent claims require multitag amplification and tagging of a 16S rRNA or 18S rRNA variable region for each sample, pooling followed by sequencing-by-synthesis, demultiplexing by tag sequence identity, and determining microbial community profiles by aligning reads or subsequences to microbial rRNA reference sequences.
Stated Advantages
Enables determining microbial community profiles across samples using tag identification together with alignment to microbial rRNA reference sequences.
Supports determining sequences without gel electrophoresis, Southern blot, or size separation.
Supports quantifying variant abundances via normalized counts.
Documented Applications
Microbial community profiling in environmental samples.
Clinical profiling relevant to inflammatory bowel disease, including ulcerative colitis and Crohn’s disease.
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