MULTITAG SEQUENCING ECOGENOMICS ANALYSIS-US
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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.
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Citations
42 Claims
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1-26. -26. (canceled)
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27. A kit comprising at least five pairs of tagged forward and reverse primer pairs, the pairs disposed separately, and each forward and reverse primer comprising, in 5′
- to 3′
order;
a sequence for immobilization and/or amplification, a tag sequence, and a priming sequence targeting a variable genetic region for amplification, wherein;(A) the sequence for immobilization and/or amplification is the same between said primers; (B) the tag sequence in each forward and reverse primer pair is the same, and different from the tag sequence of the other primer pairs; and (C) the priming sequences between the primer pairs are the same, and target for amplification a variable genetic region selected from;
a 16S rRNA sequence, an 18S rRNA sequence, an ITS sequence, a mitochondrial sequence, a microsatellite sequence, a metabolic enzyme sequence, and a variable genetic disease sequence. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
- to 3′
Specification