Multitag sequencing ecogenomics analysis-us
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Publication Number
US-9453262-B2
Publication Date
2016-09-27
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 multitag, multiplex massively-parallel polynucleotide sequencing in which amplified molecules from multiple samples are tagged with sample-specific tag sequences and then pooled for sequencing. The mixture is sequenced without gel electrophoresis, size separation of primer extension products, Southern blot transfer, or other size-separating steps, and computational methods are used to recover sample-specific sequence profiles from mixed reads by identifying the tags.
A kit is described that includes at least five pairs of tagged forward and reverse primer pairs and a software program. Each forward and reverse primer pair includes, in 5′ to 3′ order, a priming sequence, a tag sequence of from 4 to 36 nucleotides, and a probe sequence targeting a variable genetic region, where the priming sequence is shared across the primer pairs, the tag sequence is shared within a primer pair but distinct across primer pairs, and the probe sequences are the same and target variable genetic regions selected from 16S rRNA, 18S rRNA, and ITS.
The software program deconvolves, from a mixture of nucleotide sequences, the profile of nucleotide sequences for a plurality of samples by identifying the tags in the sequences. The workflow also supports normalization of variant or component abundance by total reads and contrasts with prior indexing approaches where tags or primers can distort component distributions through sequencing biases and adapter ligation artifacts.
Claims Coverage
Independent claim clm-00001 covers a kit and associated software that multiplexes at least five samples using separately disposed tagged forward and reverse primer pairs with shared priming and probe sequences, distinct tag sequences per primer pair, and tag-based computational deconvolution of pooled sequencing reads. The independent claim includes one inventive concept: tag-defined deconvolution of mixed reads into per-sample nucleotide sequence profiles targeting selected variable genetic regions (16S rRNA, 18S rRNA, or ITS).
Tagged primer pairs for multiplexing multiple samples
A kit comprising at least five pairs of tagged forward and reverse primer pairs, each primer pair disposed separately, wherein each forward and reverse primer comprises, in 5′ to 3′ order, a priming sequence, a tag sequence of from 4 to 36 nucleotides, and a probe sequence targeting a variable genetic region for amplification.
Shared priming and probe sequences with distinct tag sequences per primer pair
The priming sequence is the same between the primer pairs, the tag sequence in each forward and reverse primer pair is the same and different from the tag sequence of other primer pairs, and the probe sequences between the primer pairs are the same and target variable genetic regions selected from a 16S rRNA sequence, an 18S rRNA sequence, and an ITS sequence.
Software deconvolves pooled reads by identifying tags
A software program that deconvolves, from a mixture of nucleotide sequences, the profile of nucleotide sequences for a plurality of samples by identifying said tags in the sequences.
The independent claim is directed to multiplex polynucleotide sequencing using separately disposed tagged forward and reverse primer pairs combined with software that identifies tag sequences in mixed reads to generate per-sample nucleotide sequence profiles for variable genetic regions selected from 16S rRNA, 18S rRNA, and ITS.
Stated Advantages
Higher throughput by enabling multiplex massively-parallel polynucleotide sequencing and pooling of tagged amplified molecules for sequencing without gel electrophoresis or size separation steps.
Reduced need for cloning and fingerprinting, as sample-specific profiles are obtained computationally by tag-based deconvolution.
Normalization of variant or component abundance by total reads.
Avoidance of distribution distortion associated with prior indexing approaches that can distort component distributions.
Documented Applications
Ecogenomics analysis of microbial community samples using variable genetic regions including 16S rRNA, 18S rRNA, and ITS.
Analysis in contexts relating to dysbiosis and inflammatory bowel disease, including Crohn’s disease and ulcerative colitis.
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