title: Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing.
abstract: The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.
authors.affiliation: Department of Pathology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Boyd
authors.forename: Eleanor L
authors.initials: EL
authors.lastname: Marshall
authors.forename: Jason D
authors.initials: JD
authors.lastname: Merker
authors.forename: Jay M
authors.initials: JM
authors.lastname: Maniar
authors.forename: Lyndon N
authors.initials: LN
authors.lastname: Zhang
authors.forename: Bita
authors.initials: B
authors.lastname: Sahaf
authors.forename: Carol D
authors.initials: CD
authors.lastname: Jones
authors.forename: Birgitte B
authors.initials: BB
authors.lastname: Simen
authors.forename: Bozena
authors.initials: B
authors.lastname: Hanczaruk
authors.forename: Khoa D
authors.initials: KD
authors.lastname: Nguyen
authors.forename: Kari C
authors.initials: KC
authors.lastname: Nadeau
authors.forename: Michael
authors.initials: M
authors.lastname: Egholm
authors.forename: David B
authors.initials: DB
authors.lastname: Miklos
authors.forename: James L
authors.initials: JL
authors.lastname: Zehnder
authors.forename: Andrew Z
authors.initials: AZ
authors.lastname: Fire
mesh_terms: D000328:Adult; D000368:Aged; D001402:B-Lymphocytes / Q000276:immunology*; D016022:Case-Control Studies; D002999:Clone Cells; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D015322:Gene Rearrangement, B-Lymphocyte; D050438:Genes, Immunoglobulin Heavy Chain; D019337:Hematologic Neoplasms / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D057166:High-Throughput Screening Assays; D006801:Humans; D008875:Middle Aged; D018365:Neoplasm, Residual; D011971:Receptors, Immunologic / Q000235:genetics*; D012016:Reference Values; D017422:Sequence Analysis, DNA / Q000379:methods*; D016896:Treatment Outcome; D055815:Young Adult
chemical_list: D017931:DNA Primers; D011971:Receptors, Immunologic