System and apparatus for sequential processing of analytes
An apparatus and system are provided for simultaneously analyzing a plurality of analytes anchored to microparticles. Microparticles each having a uniform population of a single kind of analyte attached are disposed as a substantially immobilized planar array inside of a flow chamber where steps of an analytical process are carried out by delivering a sequence of processing reagents to the microparticles by a fluidic system under microprocessor control. In response to such process steps, an optical signal is generated at the surface of each microparticle which is characteristic of the interaction between the analyte carried by the microparticle and the delivered processing reagent. The plurality of analytes are simultaneously analyzed by collecting and recording images of the optical signals generated by all the microparticles in the planar array. A key feature of the invention is the correlation of the sequence of optical signals generated by each microparticle in the planar array during the analytical process.
1. A method of sequencing multiple target polynucleotides in parallel using a sequencing system, comprising:
providing a flow chamber comprising a plurality of ridges within the flow chamber running parallel to the axis of the flow chamber, the flow chamber comprising multiple microparticles immobilized between the ridges, each microparticle of the multiple microparticles including a target polynucleotide to be sequenced immobilized on the microparticle, the average distance between centers of adjacent microparticles being less than two microparticle diameters, and the ridges being of a height less than a diameter of a microparticle and of a length greater than a plurality of said diameters;
providing an oligonucleotide through an inlet of the flow chamber;
ligating the oligonucleotide to a strand hybridized to a polynucleotide of the plurality of target polynucleotides when the oligonucleotide is complementary to a single-stranded portion of the polynucleotide;
obtaining data that provides information about the plurality of target polynucleotides based on the ligation of the oligonucleotides, the information produced being governed by at least a portion of a sequence of the plurality of target polynucleotides; and
cleaving at least a portion of the oligonucleotide that was ligated.
2. The method of claim 1 , further comprising:
providing a second oligonucleotide through the inlet of a flow chamber after the cleaving of the at least a portion of the oligonucleotide that was ligated;
ligating the second oligonucleotide to the strand hybridized to a polynucleotide of the plurality of target polynucleotides when the oligonucleotide is complementary to a second single-stranded portion of the polynucleotide; and
obtaining data that provides information about the plurality of target polynucleotides based on the ligation of the second oligonucleotide, the information produced being governed by at least a portion of a sequence of the plurality of target polynucleotides.
3. The method of claim 2 , further comprising:
cleaving at least a portion of the second oligonucleotide that was ligated;
providing a third oligonucleotide through the inlet of a flow chamber after the cleaving of the at least a portion of the second oligonucleotide that was ligated;
ligating the third oligonucleotide to the strand hybridized to a polynucleotide of the plurality of target polynucleotides when the third oligonucleotide is complementary to a third single-stranded portion of the polynucleotide; and
obtaining data that provides information about the plurality of target polynucleotides based on the ligation of the third oligonucleotide, the information produced being governed by at least a portion of a sequence of the plurality of target polynucleotides.
4. The method of claim 2 , wherein
obtaining data based on the ligation of the oligonucleotide comprises obtaining data governed by a plurality of bases of a sequence of the plurality of target polynucleotides based on the ligation of the oligonucleotide; and
obtaining data based on the ligation of the second oligonucleotide comprises obtaining data governed by a plurality of bases of a sequence of the plurality of target polynucleotides based on the ligation of the second oligonucleotide.
5. The method of claim 2 , further comprising chemically constraining the microparticles in the flow chamber.
6. The method of claim 2 , wherein the data from multiple rounds of ligation comprises data indicative of which of a plurality of labels is present for a particular polynucleotide, wherein the label provides information about that particular polynucleotide.
7. The method of claim 6 , wherein the rounds of ligation that provide the data are separated by a step providing cleavage of a probe ligated in a previous round of ligation.
8. The method of claim 1 , wherein said ligating comprises providing a ligating reagent and wherein said cleaving step comprises providing a cleaving reagent.
9. The method of claim 8 , wherein the ligating reagent comprises a ligase.
10. The method of claim 8 , wherein the cleaving reagent comprises a restriction enzyme.
11. The method of claim 1 , wherein said obtaining data comprises using a data processor configured to receive data from multiple rounds of ligation of labeled probes to provide sequence data for the polynucleotides immobilized on the multiple microparticles.
12. The method of claim 11 , wherein the data processor is further configured to track each microparticle through multiple rounds of label identification.
13. The method of claim 11 , wherein the data processor is configured to obtain data that provides information that is dictated by more than one base for each polynucleotide immobilized on the multiple microparticles from each round of ligation and label identification.
14. The method of claim 11 , wherein the data processor is configured to receive data corresponding to the polynucleotides as they are processed in parallel.
15. The method of claim 1 , wherein the number of microparticles per unit area in the planar array is at least eighty percent of the number of microparticles in a hexagonal array of equal area.
16. The method of claim 1 , wherein the ridges are spaced such that the microparticles are arranged single-file between the ridges.
17. The method of claim 1 , wherein multiple ridges in the flow chamber are parallel to each other.
18. The method of claim 1 , wherein a distance between ridges is configured to allow for maximal packing of microparticles.
19. The method of claim 1 , wherein a diameter of each of the multiple microparticles is approximately a same diameter.
20. The method of claim 1 , wherein the multiple microparticles are chemically constrained in the flow chamber.
21. The method of claim 20 , wherein the multiple microparticles are constrained by a crosslinked chemical attachment.
22. The method of claim 1 , wherein said obtaining data comprises using a light source configured to provide an excitation beam and an optics system configured to receive light produced in response to the excitation beam.
23. The method of claim 1 , wherein the sequencing system further comprises a detector configured to receive, from each round of ligation and identification, data indicative of an identity of more than one base of a sequence of multiple analytes.
24. The method of claim 1 , wherein the sequencing system comprises a body attached to a plate to form the flow chamber.
25. The method of claim 1 , wherein the multiple microparticles are connected to the flow chamber in a monolayer between the ridges, said monolayer comprising multiple tiles and a detector is configured to separately generate data based on a first subset of the multiple microparticles and a second subset of the multiple microparticles physically separate from the first subset of the multiple microparticles.
26. The method of claim 1 , wherein at least 500,000 microparticles are immobilized in the flow chamber, each of the 500,000 microparticles comprising an analyte to be sequenced immobilized on the microparticle.
27. A method of sequencing multiple target polynucleotides in parallel using a sequencing system, comprising:
providing an oligonucleotide through an inlet of a flow chamber, said flow chamber comprising multiple microparticles immobilized between ridges within the flow chamber running parallel to the axis of the flow chamber, wherein said multiple microparticles include multiple target polynucleotides to be sequenced, wherein the average distance between centers of adjacent microparticles is less than two microparticle diameters, and wherein the ridges are of a height less than a diameter of a microparticle and of a length greater than a plurality of said diameters; and
sequencing at least a portion of more than one of said multiple target polynucleotides.
28. The method of claim 27 , wherein the distance between the elongated ridges having multiple microparticles immobilized therebetween is greater than the diameter of a microparticle.
29. The method of claim 27 , wherein the microparticles comprise a hexagonal array.