Immuno-PETE

A method for enriching from a sample a plurality of structurally different target polynucleotides, wherein individual target polynucleotides of the plurality comprise immune cell receptor V, J, and C and optionally D gene regions, the method comprising:

• a) providing a reaction mixture comprising:
• i) the plurality of structurally different target polynucleotides;
• and
• ii) a plurality of immune cell receptor C gene specific primers, wherein the plurality of immune cell receptor C gene specific primers having the following regions from 5′ to 3′:
• [5′-Phos], [SPLINT], [BARCODE], and
• [FW], wherein:
• [5′-Phos] comprises a 5′ phosphate;
• [SPLINT] comprises an adaptor hybridization site of 2-8 nucleotides in length;
• [BARCODE] comprises a barcode region of at least 6 nucleotides in length, wherein each nucleotide of the barcode region is independently selected from the group consisting of N and W;
• and
• [FW] of each immune cell receptor C gene specific primer comprises a structurally distinct region that specifically hybridizes to an immune cell receptor C gene,
• wherein the immune cell receptor C gene specific primers are hybridized to the C gene regions of the target polynucleotides;
• b) extending the hybridized immune cell receptor C gene specific primers with a polymerase, and then removing un-extended immune cell receptor C gene specific primers, if present, wherein the extended immune cell receptor C gene specific primers comprise at least a portion of the immune cell receptor C region, optionally the immune cell receptor D region, at least a portion of the immune cell receptor J region, and at least a portion of the immune cell receptor V region;
• c) hybridizing a first universal adaptor to the [SPLINT] adaptor hybridization site of the extended immune cell receptor C gene specific primers, wherein said first universal adaptor is a double-stranded adaptor comprising a single-stranded overhang region that hybridizes to the [SPLINT] adaptor hybridization site of said extended primers;
• d) ligating the hybridized first universal adapters to the extended immune cell receptor C gene specific primers, and then removing un-ligated adapters, if present;
• e) hybridizing a plurality of immune cell receptor V gene specific primers to the V region portions of the extended immune cell receptor C gene specific primers, wherein the immune cell receptor V gene specific primers comprise a 3′ V gene hybridizing region and a 5′ second universal adapter region;
• and
• f) extending the hybridized immune cell receptor V gene specific primers with a polymerase, thereby forming a plurality of structurally different double-stranded products, each comprising at least a portion of the immune cell receptor V region, optionally the immune cell receptor D region, and at least a portion of the immune cell C region flanked by a first and second universal adapter sequence.