Multitag sequencing ecogenomics analysis-us
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.
1. A kit comprising at least five pairs of tagged forward and reverse primer pairs, and a software program:
wherein the primer pairs are disposed separately, and each forward and reverse primer comprises, in 5′ to 3′ order: a priming sequence, a tag sequence of from 4 to 36 nucleotides in length, and a probe sequence targeting a variable genetic region for amplification, wherein:
(A) the priming sequence is the same between said primer pairs, with the proviso that forward and reverse primers may have the same or different priming sequences;
(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 probe 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, and an ITS sequence;
and wherein the software program deconvolves, from a mixture of nucleotide sequences, the profile of nucleotide sequences for a plurality of samples by identifying said tags in the sequences.
2. The kit of claim 1 , wherein the variable genetic region varies between species.
3. The kit of claim 1 , wherein the variable genetic region varies within a species.
4. The kit of claim 1 , further comprising a primer complementary to and effective for elongation from said priming sequence.
5. The kit of claim 1 , wherein the kit comprises a set of from 10 to 25 tagged primer pairs.
6. The kit of claim 1 , wherein the kit comprises from 20 to 50 tagged primer pairs.
7. The kit of claim 1 , wherein the kit comprises from 50 to 150 tagged primers pairs.
8. The kit of claim 1 , wherein the kit comprises from 100 to 500 tagged primers pairs.
9. The kit of claim 1 , wherein the tag sequences are 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
10. The kit of claim 1 , 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.
11. The kit of claim 1 , wherein the software is implemented in C, C++, JAVA, Fortran, or Basic.
12. The kit of claim 1 , wherein the software is implemented as a PERL script.
13. The kit of claim 1 , 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 tags for the associate array,
associate each tag 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.
14. The kit of claim 1 , wherein the primer pairs are disposed in separate wells of a titer plate.