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US 2021/0172015 A1Published application
Immune profiling by primer extension target enrichment
AssigneeRoche Sequencing Solutions, Inc.
InventorsJan Berka, Carolina Dallett, Richard Dannebaum, Brian Godwin, Seoyoung Kim, Jainee Lewis, Khai Luong, Sedide Ozturk, Joseph Platzer, Florian Rubelt, Dilduz Telman
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Abstract
Methods and compositions are described herein for primer extension target enrichment of immune receptor (BCR or TCR) sequences.
Claims38
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.
15 more elements
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 [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;
- 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%.
18 more elements
Dependencies are inferred from the claim text.