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 generating a library of enriched single cell RNA transcripts comprising:
(a) preparing from a plurality of single cells a barcoded single cell whole transcriptome amplification (WTA) product comprising a plurality of cDNAs, wherein each cDNA comprises a transcript sequence, a unique molecular identifier (UMI), and a cell barcode (CB);
(b) enriching cDNAs specific to one or more transcripts of interest from the WTA product;
(c) replicating the enriched cDNAs by performing primer extension of the enriched cDNAs with one or more primers targeting one or more regions of the one or more transcripts of interest, wherein the 5′ edge of the transcript sequences are set;
(d) amplifying the primer extension products.
2 . The method of claim 1 , further comprising sequencing the library with sequencing primers that are complementary to a sequence within the transcript of interest and a primer for sequencing the UMI and CB, whereby the sequences can be assigned to single cells.
3 . The method of claim 1 , further comprising generating a whole transcriptome 3′ sequencing library from the WTA product and sequencing the library, whereby paired transcript of interest sequences and full transcriptome sequences are obtained for the same single cells.
4 . The method of claim 1 , further comprising an amplification step after step (b) and before step (c).
5 . The method of claim 1 , wherein enriching cDNAs specific to a transcript of interest comprises:
(a) hybridizing the cDNAs specific to a transcript of interest to tagged oligonucleotides specific to a transcript of interest and binding to a capture bead specific for the tag or wherein the tagged oligonucleotides are already bound to beads; or
(b) amplifying the cDNAs specific to a transcript of interest using a tagged 5′ primer complementary to a specific transcript and a 3′ primer and enriching the amplification product by binding the tag introduced by the 5′ primer to a capture bead specific for the tag.
6 . The method of claim 1 , wherein any amplification step or primer extension step adds a universal sequencing adapter to the cDNA.
7 . The method of claim 1 , further comprising circularization of the primer extension products in step (c), comprising:
(a) amplifying the primer extension products using primers comprising complementary sequences in the 5′ ends followed by a deoxy-uracil residue (dU); and
(b) providing a uracil-specific excision enzyme and T4 ligase to cleave the dU residues and generate complementary sticky ends that mediate circularization and ligation, whereby the CB and the 5′ edge of the transcript sequence are within 100 bases of each other in the circularized cDNA.
8 . The method of claim 1 , wherein the single cells comprise T cells, B cells, and/or NK cells.
9 . The method of claim 1 , wherein the transcript of interest is in a T cell receptor (TCR), a B cell receptor (BCR), or chimeric antigen receptor (CAR) sequence.
10 . The method of claim 1 , wherein the transcript of interest is a variable region of a TCR, BCR, or CAR.
11 . The method of claim 1 , wherein the one or more regions of the transcript of interest targeted in primer extension in step (c) are one or more variable regions of a TCR, BCR, or CAR.14.
12 . A library of enriched single cell RNA transcripts obtained by the method of claim 1 .
13 . The library of claim 12 , wherein the CB is within 100 bases of the transcript of interest in the cDNA of step (d), wherein at least a subset of the cDNAs from the WTA product comprise transcripts of interest that were greater than 1 kb away from the CB.