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 multiplex method for determining abundance profiles of one or more target polynucleotide sequences across a plurality of samples, comprising:
amplifying and tagging target polynucleotides by PCR in each of said plurality of samples with an amplification primer comprising a high throughput sequencing adaptor, a sample-specific tag sequence of at least four nucleotides in length, and a priming sequence to amplify said target polynucleotide sequence(s);
combining the amplified polynucleotides and sequencing the polynucleotide pool in high throughput, so as to determine the sequence of at least 300 tagged polynucleotides for each of said samples;
assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the sample-specific tag, thereby determining abundance profiles of the target polynucleotide sequence(s) across the samples.
2. The method of claim 1 , wherein the samples are clinical or environmental microbial community samples.
3. The method of claim 2 , wherein the samples are clinical samples for patients suspected of having Inflammatory Bowel Disease (IBD), Crohn's Disease (CD), or ulcerative colitis (UC).
4. The method of claim 1 , wherein the samples are clinical, agricultural, food, biosecurity, veterinary, ecological, zoological, or oceanological samples.
5. The method of claim 4 , wherein the samples are environmental samples selected from soil and water.
6. The method of claim 1 , wherein the target polynucleotides are bacterial genomic DNA, Archae genomic DNA, fungal genomic DNA, eukaryotic genomic DNA, chloroplast DNA, or mitochondrial DNA.
7. The method of claim 1 , wherein the target polynucleotides are cDNA.
8. The method of claim 6 , wherein the priming sequence hybridizes to a target sequence that is 3′ to a variable genetic region.
9. The method of claim 8 , wherein the variable genetic region varies across species.
10. The method of claim 9 , wherein the variable genetic region is a variable 16S rRNA sequence, a variable 18S rRNA sequence, or a variable rRNA ITS sequence.
11. The method of claim 9 , wherein the variable genetic region is a mitochondrial sequence, microsatellite sequence, or metabolic enzyme sequence.
12. The method of claim 1 , wherein the number of samples is at least 10.
13. The method of claim 1 , wherein the number of samples is at least 25.
14. The method of claim 1 , wherein the number of samples is at least 50.
15. The method of claim 1 , wherein the sample-specific tags are each from 4 to 25 nucleotides in length.
16. The method of claim 15 , wherein the sample-specific tags are 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
17. The method of claim 15 , wherein the sample-specific tag sequences do not include any homodinucleotide sequence.
18. The method of claim 17 , wherein no two of the tag sequences are complementary.
19. The method of claim 1 , wherein the priming sequence is the same across samples.
20. The method of claim 1 , wherein the abundance of the target polynucleotide is normalized across the samples.
21. The method of claim 1 , wherein the sequencing adaptor immobilizes the individual polynucleotides for clonal amplification and sequencing.
22. The method of claim 21 , wherein the sequencing is pyrosequencing.
23. The method of claim 21 , wherein the sequencing is sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-hybridization.
24. The method of claim 1 , wherein the sequencing is single molecule sequencing.
25. The method of claim 1 , wherein the PCR amplifications employ a forward and reverse primer that both comprise the high throughput sequencing adaptor and the sample-specific tag.
26. A method for determining microbial community profiles across a plurality of clinical samples or a plurality of environmental samples, comprising:
amplifying and tagging target polynucleotides by PCR in each of said plurality of samples with an amplification primer pair comprising:
a high throughput sequencing adaptor,
a tag sequence to identify the sample, and which is different by at least two nucleotides across the samples, and
a priming sequence that is the same in each sample, the priming sequence hybridizing 3′ to a genetic region that is variable across microbial species;
combining the amplified polynucleotides and sequencing the polynucleotide pool in high throughput, so as to determine the sequence of at least 300 tagged polynucleotides for each of said samples;
assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the tag, thereby determining microbial community profiles across the samples.