# US 2014/0194323 A1 · Multitag sequencing ecogenomics analysis-us

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- Number: US 2014/0194323 A1
- Document ID: US20140194323A1
- Status: Published application
- Kind: A1
- Application number: 14/066,967
- Filed: 2013-10-30
- Published: 2014-07-10
- Earliest claimed: 2006-11-15
- Assignees: BioSpherex, LLC, A Limited Liability Company
- Applicants: BioSpherex, LLC a Limited Liability Company
- Inventors: Patrick M. Gillevet
- Web page: https://patentgrep.com/patent/US20140194323A1

## CPC

- C12Q 1/689: for bacteria
- C12Q 1/6858: Allele-specific amplification
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim at position 1

1-26. (canceled)

### Claim 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.

### Claim 28 (depends on 27)

The kit of claim 27, wherein the variable genetic region varies between species.

### Claim 29 (depends on 27)

The kit of claim 27, wherein the variable genetic region varies within a species.

### Claim 30 (depends on 27)

The kit of claim 27, further comprising a primer complementary to and effective for elongation from said sequence for immobilization and/or amplification.

### Claim 31 (depends on 27)

The kit of claim 27, wherein the kit comprises a set of from 10 to 25 tagged primer pairs.

### Claim 32 (depends on 27)

The kit of claim 27, wherein the kit comprises from 20 to 50 tagged primer pairs.

### Claim 33 (depends on 27)

The kit of claim 27, wherein the kit comprises from 50 to 150 tagged primers pairs.

### Claim 34 (depends on 27)

The kit of claim 27, wherein the kit comprises from 100 to 500 tagged primers pairs.

### Claim 35 (depends on 27)

The kit of claim 27, wherein the priming sequence is complementary to a 3′ variable genetic region selected from a 16S rRNA sequence, an 18S rRNA sequence, and ITS sequence.

### Claim 36 (depends on 27)

The kit of claim 27, wherein the tag sequences are 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.

### Claim 37 (depends on 27)

The kit of claim 27, wherein each tag sequence is 4 to 36 nucleotides in length.

### Claim 38 (depends on 27)

The kit of claim 27, wherein the tag sequence is not the complementary sequence to any other tag sequence; the tag sequence does not contain any homodinucleotide sequences; and the junction sequence, if any, between the tag sequence and the adjacent parts of the primer is not a homodinucleotide sequence.

### Claim 39 (depends on 27)

The kit of claim 27, further comprising a software program for deconvolving, from a mixture of sequences, the sequences of each sample in the mixture by identifying sample-specific tags in sequences.

### Claim 40 (depends on 39)

The kit of claim 39, wherein the algorithm is implemented in C, C++, JAVA, Fortran, or Basic.

### Claim 41 (depends on 39)

The kit of claim 39, wherein the algorithm is implemented as a PERL script.

### Claim 42 (depends on 39)

The kit of claim 39, wherein the software program:
reads all sequence reads,
searches the beginning of each sequence read and identifies the tag,
builds an associative array linking tag and sequence reads,
sorts the keys for the associate array,
associate each key with the corresponding sample,
pool all sequence reads for each sample,
analyze each sample separately,
normalize the abundance of each component within each sample with respect to the total reads within that sample.