T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

The allele frequency spectrum of polymorphisms in DNA sequences can be used to test for signatures of natural selection that depart from the expected frequency spectrum under the neutral theory. We observed a significant (P = 0.001) correlation between the Tajima's D test statistic in full resequencing data and Tajima's D in a dense, genome-wide data set of genotyped polymorphisms for a set of 179 genes. Based on this, we used a sliding window analysis of Tajima's D across the human genome to identify regions putatively subject to strong, recent, selective sweeps. This survey identified seven Contiguous Regions of Tajima's D Reduction (CRTRs) in an African-descent population (AD), 23 in a European-descent population (ED), and 29 in a Chinese-descent population (XD). Only four CRTRs overlapped between populations: three between ED and XD and one between AD and ED. Full resequencing of eight genes within six CRTRs demonstrated frequency spectra inconsistent with neutral expectations for at least one gene within each CRTR. Identification of the functional polymorphism (and/or haplotype) responsible for the selective sweeps within each CRTR may provide interesting insights into the strongest selective pressures experienced by the human genome over recent evolutionary history.

To promote the clinical and epidemiological studies that improve our understanding of human genetic susceptibility to environmental exposure, the Environmental Genome Project (EGP) has scanned 213 environmental response genes involved in DNA repair, cell cycle regulation, apoptosis, and metabolism for single nucleotide polymorphisms (SNPs). Many of these genes have been implicated by loss-of-function mutations associated with severe diseases attributable to decreased protection of genomic integrity. Therefore, the hypothesis for these studies is that individuals with functionally significant polymorphisms within these genes may be particularly susceptible to genotoxic environmental agents. On average, 20.4 kb of baseline genomic sequence or 86% of each gene, including a substantial amount of introns, all exons, and 1.3 kb upstream and downstream, were scanned for variations in the 90 samples of the Polymorphism Discovery Resource panel. The average nucleotide diversity across the 4.2 MB of these 213 genes is 6.7 × 10-4, or one SNP every 1500 bp, when two random chromosomes are compared. The average candidate environmental response gene contains 26 PHASE inferred haplotypes, 34 common SNPs, 6.2 coding SNPs (cSNPs), and 2.5 nonsynonymous cSNPs. SIFT and Polyphen analysis of 541 nonsynonymous cSNPs identified 57 potentially deleterious SNPs. An additional eight polymorphisms predict altered protein translation. Because these genes represent 1% of all known human genes, extrapolation from these data predicts the total genomic set of cSNPs, nonsynonymous cSNPs, and potentially deleterious nonsynonymous cSNPs. The implications for the use of these data in direct and indirect association studies of environmentally induced diseases are discussed

Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. The precision of the assay is demonstrated across five orders of magnitude. The accuracy for each clone is within an overall factor of three across the 100,000 fold dynamic range. Additionally, the assay is shown to be highly repeatable.

The two main lineages of T lymphocytes develop from multi potent precursors in the human thymus. The most common type in blood are αβ T cells, which bind to antigenic peptides displayed on the surface of cells by human leukocyte antigen (HLA) molecules. Far less well understood are γδ T cells, which do not bind HLA: peptide complexes and are more prevalent in the gut mucosa. For both lineages, their ability to recognize a diverse array of antigens is mediated by a rearranged Y-like receptor on their surface, the T cell receptor (TCR), composed and of an α and β chain for αβ T cells or a γ and δ chain for γδ T cells. The canonical model for commitment from the precursor to one these two lineages assumes that γ, δ, and β chains rearrange prior to commitment to αβ or γδ T cells. A crucial step towards better understanding the role of γδ T cells is to work out the developmental process. To test the standard model and to understand the γδ TCR repertoire, we use high-throughput sequencing to catalog millions of TCRγ and TCRβ chains from peripheral blood αβ and γδ T cells, from three unrelated individuals. Almost all sampled αβ and γδ T cells have rearranged TCRγ sequences. While sampled αβ T cells have a diverse repertoire of rearranged TCRβ chains, less than 10% of γδ T cells in peripheral blood have a rearranged TCRβ chain. Our data indicate that TCRγ rearranges in all T lymphocytes, consistent with TCRγ rearranging prior to T cell lineage commitment, while rearrangement of the TCRβ locus is restricted, and occurs after T cell precursors commit to the αβ T cell lineage. This result explains the conundrum in T cell leukemia and lymphoma that TCRγ is almost always rearranged and TCRβ is only rearranged in a subset of cancers. As high-throughput sequencing of TCRs is translated into the clinic for monitoring minimal residual for leukemia/lymphoma, our data suggests the sequencing target needs to be TCR γ.