Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries
The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.
1. A method of distinguishing cells by genotype comprising:
(a) constructing a library comprising a plurality of nucleic acids wherein each nucleic acid comprises a gene comprising a polyA tail, a unique molecular identifier (UMI) and a cell barcode (cell BC) flanked by sequencing adapters at the 5′ and 3′ end,
(b) amplifying each nucleic acid in the library to create a first PCR product using a tagged 5′ primer comprising a binding site for a second PCR product and a sequence complementary to a specific gene of interest and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating a first PCR product,
(c) selectively enriching the first PCR product by binding to the tag introduced by the 5′ primer or a targeted 3′ capture with a bifunctional bead or targeted capture bead,
(d) amplifying the tag-enriched first PCR product with a 5′ primer comprising the binding site for the second PCR product and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating a second PCR product,
(e) optionally amplifying the second PCR product with a 5′ primer comprising the binding site for a third PCR product and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating the third PCR product, and
(f) determining the genotype of the cell by identifying the UMI and cell BC, thereby distinguishing the cells by genotype.
2. The method of claim 1 , further comprising size selecting a final product comprising the specific gene of interest.
3. The method of claim 1 , wherein the sequencing adapters are switching mechanism at 5′ end of RNA template (SMART) sequences at the 5′ and 3′ end.
4. The method of claim 1 , wherein the binding site for the second PCR product is an oligomer comprising a primer binding site for sequencing.
5. The method of claim 1 , wherein the amplifying the second PCR product is performed to generate a third PCR product, and further comprising sequencing the third PCR product by third generation sequencing.
6. The method of claim 5 , further comprising sequencing the second PCR product by next generation sequencing.
7. The method of claim 4 , wherein the binding site is an oligomer comprising a P7 sequence.
8. The method of claim 1 , wherein the 5′ primer comprising the binding site for the second PCR product to amplify the first PCR product further comprises a sequence to bind a flow cell.
9. The method of claim 8 , wherein the 5′ primer comprising the binding site for the second PCR product to amplify the first PCR product further comprises a sequence allowing multiple sequencing libraries to be sequenced simultaneously.
10. The method of claim 8 , wherein the 5′ primer comprising the binding site for the second PCR product to amplify the first PCR product further comprises a sequence providing an additional primer binding site.
11. The method of claim 8 , wherein the sequence to bind a flow cell is a P7 sequence and the flow cell surface comprises oligos complementary to the P7 sequence.
12. The method of claim 9 , wherein the sequence allowing multiple sequencing libraries to be sequenced simultaneously is a library barcode.
13. The method of claim 10 , wherein the 5′ primer comprising the binding site for the second PCR product to amplify the first PCR product further comprises a sequence comprising a primer binding site for a Read2 sequencing primer.
14. The method of claim 3 , wherein the 3′ primer complementary to the SMART sequence at the 3′ end of the nucleic acid to amplify the first PCR product further comprises a sequence providing an additional primer binding site, optionally wherein the sequence providing an additional primer binding site is a custom read1 primer binding site (CR1P).
15. The method of claim 3 , wherein the primer complementary to the SMART sequence at the 3′ end of the nucleic acid to amplify the first PCR product further comprises a sequence to allow fragments to bind a flowcell, optionally wherein the sequence to allow fragments to bind a flowcell is a P5 sequence.
16. The method of claim 1 , wherein the specific gene of interest comprises a mutation, deletion, insertion, translocation, single nucleotide polymorphism (SNP), splice variant or any combination thereof associated with a particular attribute in the specific gene of interest.
17. The method of claim 1 , wherein the specific gene of interest is a cancer gene, a tumor protein P53 gene, a KIAA1549:BRAF fusion gene, or an acute myeloid leukemia (AML) gene.
18. The method of claim 17 , wherein the AML gene is a DNA methyltransferase gene, optionally wherein the DNA methyltransferase is DNA 5-cytosine methyltransferase 3a (DNMT3A).
19. The method of claim 1 , wherein the tagged 5′ primer comprises a biotin tag.
20. The method of claim 1 , wherein the tagged 5′ primer and the 3′ primer further comprise complementary sequences on 5′ ends of the primers followed by a deoxy-uracil residue, thereby generating a first PCR product comprising the complementary sequences,
and further comprising
(a) treating the first PCR product with a uracil-specific excision reagent enzyme,
(b) circularizing the first PCR product by sticky end ligation, and
(c) amplifying the tag-enriched circularized PCR product with a 5′ primer complementary to the gene of interest and having a sequence adapter and a 3′ primer having a polyA tail and another sequence adapter thereby generating the second PCR product.
21. The method of claim 16 , wherein the mutation is within 1 kilobase of the polyA tail or wherein the mutation is anywhere in the gene.