US2024026570A1PendingUtilityA1

Immune profiling by primer extension target enrichment

76
Assignee: ROCHE SEQUENCING SOLUTIONS INCPriority: Jun 1, 2016Filed: Aug 14, 2023Published: Jan 25, 2024
Est. expiryJun 1, 2036(~9.9 yrs left)· nominal 20-yr term from priority
Inventors:Jan BerkaCarolina DallettRichard DannebaumBrian Christopher GodwinSeoyoung KimJainee LewisKhai LuongSedide OzturkJoseph PlatzerFlorian RubeltDilduz Telman
C40B 50/06C12Q 1/68C12Q 2600/16C12N 15/1065C12Q 1/6886C12Q 1/6881
76
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Claims

Abstract

Methods and compositions are described herein for primer extension target enrichment of immune receptor (BCR or TCR) sequences.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method for enriching a sample for a plurality of structurally different target polynucleotides comprising an immune gene sequence the method comprising:
 a) contacting a sample with a plurality of immune cell receptor V gene specific primers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT1], [BARCODE], and [V], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT] is a first adaptor sequence; [BARCODE] is a unique molecular identifier barcode; and [V] is a sequence capable of hybridizing to an immune cell receptor V gene;   b) hybridizing and extending the V gene specific primers to form a plurality of first double-stranded primer extension products;   c) contacting the sample with an exonuclease to remove unhybridized V gene specific primers from the first double stranded primer extension products;   d) contacting the sample with a plurality of immune cell receptor J gene specific primers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT2], and [J], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT2] is a second adaptor sequence; and [J] is a sequence capable of hybridizing to an immune cell receptor J gene; and further contacting the sample with a first universal primer capable of hybridizing to the first adaptor sequence;   e) hybridizing and extending the J gene specific primers and the first universal primer to form a plurality of second double-stranded primer extension products;   f) contacting the sample with an exonuclease to remove unhybridized J gene specific primers and first universal primer from the second double-stranded primer extension products;   g) contacting the sample with first and second universal primers capable of hybridizing to the first and second adaptor sequences;   h) amplifying the plurality of second double-stranded primer extension products thereby enriching the plurality of structurally different target polynucleotides comprising an immune gene sequence.   
     
     
         2 . The method of  claim 1 , wherein the immune genes comprise one or more of T-cell receptor alpha (TCRA), T-cell receptor beta (TCRB), T-cell receptor gamma (TCRG), T-cell receptor delta (TCRD), Immunoglobulin heavy chain (IGH), Immunoglobulin light chain-kappa (IGK) and Immunoglobulin light lambda (IGL). 
     
     
         3 . The method of  claim 1 , wherein the plurality of V gene specific primers and the plurality of J gene specific primers include primers from Table 2a. 
     
     
         4 . The method of  claim 1 , wherein hybridizing in steps b) and/or e) comprises one or more cycles of a step-wise temperature drop of two or more steps. 
     
     
         5 . The method of  claim 4 , wherein hybridizing in steps b) and/or e) comprises 20 cycles of temperature change from 60° C. to 57.5° C. and to 55° C. 
     
     
         6 . The method of  claim 1 , wherein hybridizing and extending in step e) comprises two or more cycles of duplex denaturation, primer annealing and primer extension. 
     
     
         7 . The method of  claim 6 , wherein primer annealing comprises one or more cycles of a step-wise temperature drop of two or more steps. 
     
     
         8 . The method of  claim 7 , wherein hybridizing and extending in step e) comprises 10 cycles temperature change from >90° C., to 60° C. to 57.5° C., to 55° C. and to 72° C. 
     
     
         9 . The method of  claim 1 , wherein the first and second universal primers in step g) comprise additional 5′sequences not present in the first and second adaptors. 
     
     
         10 . The method of  claim 9 , wherein the first universal primer in step d) does not comprise additional 5′sequences not present in the first adaptor. 
     
     
         11 . The method of  claim 1 , wherein the exonuclease in steps c) and/or f) is thermolabile. 
     
     
         12 . The method of  claim 11 , wherein the exonuclease is Exonuclease I. 
     
     
         13 . The method of  claim 1 , wherein extending in steps b) and/or e) is with a high-fidelity DNA polymerase. 
     
     
         14 . The method of  claim 1 , further comprising a purification step after steps c) and/or h). 
     
     
         15 . The method of  claim 1 , not having a purification step between steps f) and g). 
     
     
         16 . The method of  claim 15 , wherein the first and second universal primers comprise a modification preventing digestion of the primers with the exonuclease. 
     
     
         17 . The method of  claim 16 , wherein the primers comprise one or more nucleotides having a modification selected from: phosphorothioate (PS) bond, a 2′-O-methyl (2′OMe), a 2′-fluoride and Inverted ddT. 
     
     
         18 . The method of  claim 1 , wherein the V-gene specific primers, the J-gene specific primers and the first universal primer in step d) do not comprise a modification preventing digestion of the primers with the exonuclease. 
     
     
         19 . The method of  claim 1 , further comprising sequencing the plurality of structurally different target polynucleotides comprising an immune gene sequence. 
     
     
         20 . A method for contamination-reduced sequencing a plurality of structurally different target polynucleotides comprising an immune gene sequence the method comprising:
 a) contacting a sample with a plurality of immune cell receptor V gene specific primers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT1], [BARCODE], and [V], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT] is a first adaptor sequence; [BARCODE] is a unique molecular identifier barcode (UMI); and [V] is a sequence capable of hybridizing to an immune cell receptor V gene;   b) hybridizing and extending the V gene specific primers to form a plurality of first double-stranded primer extension products;   c) contacting the sample with an exonuclease to remove unhybridized V gene specific primers from the first double stranded primer extension products;   d) contacting the sample with a plurality of immune cell receptor J gene specific primers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT2], and [ ], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT2] is a second adaptor sequence; and [ ] is a sequence capable of hybridizing to an immune cell receptor J gene; and further contacting the sample with a first universal primer capable of hybridizing to the first adaptor sequence;   e) hybridizing and extending the J gene specific primers and the first universal primer to form a plurality of second double-stranded primer extension products;   f) contacting the sample with an exonuclease to remove unhybridized J gene specific primers and first universal primer from the second double-stranded primer extension products;   g) contacting the sample with first and second universal primers capable of hybridizing to the first and second adaptor sequences;   h) amplifying the plurality of second double-stranded primer extension products thereby enriching the plurality of structurally different target polynucleotides comprising an immune gene sequence;   i) sequencing the plurality of structurally different target polynucleotides comprising an immune gene sequence to obtain a dataset of sequence reads;   j) grouping the sequence reads having an identical UMI into UMI families;   k) removing from the dataset UMI families with relative representation of less than 10%.   
     
     
         21 . The method of  claim 20 , wherein representation in step k) is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. 
     
     
         22 . The method of  claim 20 , wherein the immune genes comprise one or more of T-cell receptor alpha (TCRA), T-cell receptor beta (TCRB), T-cell receptor gamma (TCRG), T-cell receptor delta (TCRD), Immunoglobulin heavy chain (IGH), Immunoglobulin light chain-kappa (IGK) and Immunoglobulin light chain-lambda (IGL). 
     
     
         23 . The method of  claim 20 , wherein the plurality of V gene specific primers and the plurality of J gene specific primers include primers from Table 2a. 
     
     
         24 . The method of  claim 20 , wherein hybridizing in steps b) and/or e) comprises one or more cycles of a step-wise temperature drop of two or more steps. 
     
     
         25 . The method of  claim 24 , wherein hybridizing in steps b) and/or e) comprises 20 cycles of temperature change from 60° C. to 57.5° C. and to 55° C. 
     
     
         26 . The method of  claim 20 , wherein hybridizing and extending in step e) comprises two or more cycles of duplex denaturation, primer annealing and primer extension. 
     
     
         27 . The method of  claim 26 , wherein primer annealing comprises one or more cycles of a step-wise temperature drop of two or more steps. 
     
     
         28 . The method of  claim 27 , wherein hybridizing and extending in step e) comprises 10 cycles temperature change from >90° C., to 60° C. to 57.5° C., to 55° C. and to 72° C. 
     
     
         29 . The method of  claim 20 , wherein the first and second universal primers in step g) comprise additional 5′sequences not present in the first and second adaptors. 
     
     
         30 . The method of  claim 29 , wherein the first universal primer in step d) does not comprise additional 5′sequences not present in the first adaptor. 
     
     
         31 . The method of  claim 20 , wherein the exonuclease in steps c) and/or f) is thermolabile. 
     
     
         32 . The method of  claim 31 , wherein the exonuclease is Exonuclease I. 
     
     
         33 . The method of  claim 20 , wherein extending in steps b) and/or e) is with a high-fidelity DNA polymerase. 
     
     
         34 . The method of  claim 20 , further comprising a purification step after steps c) and/or h). 
     
     
         35 . The method of  claim 20 , not having a purification step between steps f) and g). 
     
     
         36 . The method of  claim 35 , wherein the first and second universal primers comprise a modification preventing digestion of the primers with the exonuclease. 
     
     
         37 . The method of  claim 36 , wherein the primers comprise one or more nucleotides having a modification selected from: phosphorothioate (PS) bond, a 2′-O-methyl (2′OMe), a 2′-fluoride and Inverted ddT. 
     
     
         38 . The method of  claim 20 , wherein the V-gene specific primers, the J-gene specific primers and the first universal primer in step d) do not comprise a modification preventing digestion of the primers with the exonuclease.

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