Methods for analyzing genomic variation within cells
Methods, devices and systems for analyzing precious samples of cells, including single cells are provided. The methods, devices, and systems in various embodiments of the invention are used to assess genomic heterogeneity, which has been recognized as a central feature of many cancers and plays a critical role in disease initiation, progression, and response to treatment. The methods devices and systems are also used to analyze embryonic biopsies for preimplantation genetic diagnosis (PGD). In one embodiment, the devices, systems and methods provided herein allow for the construction of genomic and RNA-seq libraries without a pre-amplification step.
1. A method for analyzing genomic variation within a population of cells, the method comprising
distributing individual cells and/or individual nuclei from a cell suspension into a plurality of containers to obtain a plurality of distributed individual cells and/or individual nuclei;
creating indexed single cell sequencing libraries from the single cells and/or individual nuclei in one or more of the plurality of containers; wherein creating the indexed-single cell sequencing libraries comprises,
subjecting unamplified polynucleotides from the single cells and/or individual nuclei to a transposase reaction, wherein the transposase reaction comprises fragmenting the unamplified polynucleotides from the single cells and/or individual nuclei to generate fragmented polynucleotides, and tagging the fragmented polynucleotides with a tagging sequence to generate tagmented polynucleotides;
subjecting the tagmented polynucleotides to 9 to 15 cycles of a polymerase chain reaction (PCR) with indexed primers and sequencing adaptors, thereby generating indexed single cell sequencing libraries,
pooling a subset of the indexed single cell sequencing libraries to make a pooled library comprising genomic information of a subset of the plurality of distributed individual cells and/or individual nuclei;
sequencing the pooled library to obtain incomplete genomic information of the subset of the plurality of distributed individual cells and/or individual nuclei; and
aligning reads obtained from the sequencing of the pooled library to a reference genome in order to detect the presence or absence of genomic variation in the one or more distributed individual cells and/or individual nuclei.
2. The method of claim 1 , wherein the transposase reaction is a one-step transposase reaction.
3. The method of claim 1 , wherein from about 100 to about 1000 individual cells and/or nuclei are each distributed into individual containers.
4. The method of claim 1 , wherein the cell population is from a tumor sample.
5. The method of claim 4 , wherein the tumor sample comprises a solid tumor, resected tissue or a fine needle aspirate.
6. The method of claim 4 , wherein the tumor is a breast tumor.
7. The method of claim 1 , wherein the plurality of containers have an average volume of from 1 nL to 1000 nL or from 0.1 nL to 1 nL.
8. The method of claim 1 , wherein the plurality of containers comprises a plurality of chambers, a plurality of open microwells, or a plurality of microdroplets.
9. The method of claim 8 , wherein the plurality of chambers comprises from 100 to 10,000 chambers, or from 10,000 to 100,000 chambers.
10. The method of claim 8 , wherein from about 100 to about 1000 individual cells and/or nuclei are each distributed into individual chambers.
11. The method of claim 1 , wherein sequencing the pooled library comprises sequencing to sufficient depth to obtain an average of between 0.01% and 0.1% coverage of the genome of each cell, between 0.1% and 1% coverage of the genome of each cell, between 1% and 5% coverage of the genome of each cell, between 5% and 10% coverage of the genome of each cell, between 10% and 25% coverage of the genome of each cell, or between 25° A and 50% coverage of the genome of each cell.
12. The method of claim 1 , wherein the pooled library is sequenced to sufficient depth to obtain between 10× and 100× coverage of the average bulk genome of the population of cells.
13. The method of claim 1 , wherein the genomic variation is copy number variation, translocations, loss of heterozygosity, single nucleotide polymorphism or a combination thereof.
14. The method of claim 13 , wherein the genomic variation is copy number variation.
15. The method of claim 1 , wherein the transposase comprises Tn5 transposase.
16. The method of claim 1 , further comprising determining the phylogenic lineage of the identified subpopulations.
17. The method of claim 1 , further comprising, analyzing a distribution of genomic variation(s) across the individual cells and/or individual nuclei to identify subpopulations of single cells and/or nuclei that share common genomic features.
18. The method of claim 17 , further comprising, analyzing combined genomic features from identified subpopulations of cells and/or nuclei to identify genomic features that exist within the identified subpopulations.