Development and optimization of a hybridization technique to type the classical class I and class II B genes of the chicken MHC.

The classical class I and class II molecules of the major histocompatibility complex (MHC) play crucial roles in immune responses to infectious pathogens and vaccines as well as being important for autoimmunity, allergy, cancer and reproduction. These classical MHC genes are the most polymorphic known, with roughly 10,000 alleles in humans. In chickens, the MHC (also known as the BF-BL region) determines decisive resistance and susceptibility to infectious pathogens, but relatively few MHC alleles and haplotypes have been described in any detail. We describe a typing protocol for classical chicken class I (BF) and class II B (BLB) genes based on a hybridization method called reference strand-mediated conformational analysis (RSCA). We optimize the various steps, validate the analysis using well-characterized chicken MHC haplotypes, apply the system to type some experimental lines and discover a new chicken class I allele. This work establishes a basis for typing the MHC genes of chickens worldwide and provides an opportunity to correlate with microsatellite and with single nucleotide polymorphism (SNP) typing for approaches involving imputation.

Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE.

BACKGROUND: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. RESULTS: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. CONCLUSION: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.

Bi-Functional Chicken Immunoglobulin-Like Receptors With a Single Extracellular Domain (ChIR-AB1): Potential Framework Genes Among a Relatively Stable Number of Genes Per Haplotype.

The leukocyte receptor complex (LRC) in humans encodes many receptors with immunoglobulin-like (Ig-like) extracellular domains, including the killer Ig-like receptors (KIRs) expressed on natural killer (NK) cells among others, the leukocyte Ig-like receptors (LILRs) expressed on myeloid and B cells, and an Fc receptor (FcR), all of which have important roles in the immune response. These highly-related genes encode activating receptors with positively-charged residues in the transmembrane region, inhibitory receptors with immuno-tyrosine based motifs (ITIMs) in the cytoplasmic tail, and bi-functional receptors with both. The related chicken Ig-like receptors (ChIRs) are almost all found together on a microchromosome, with over 100 activating (A), inhibitory (B), and bi-functional (AB) genes, bearing either one or two extracellular Ig-like domains, interspersed over 500-1,000 kB in the genome of an individual chicken. Sequencing studies have suggested rapid divergence and little overlap between ChIR haplotypes, so we wished to begin to understand their genetics. We chose to use a hybridization technique, reference strand-mediated conformational analysis (RSCA), to examine the ChIR-AB1 family, with a moderate number of genes dispersed across the microchromosome. Using fluorescently-labeled references (FLR), we found that RSCA and sequencing of ChIR-AB1 extracellular exon gave two groups of peaks with mobility correlated with sequence relationship to the FLR. We used this system to examine widely-used and well-characterized experimental chicken lines, finding only one or a few simple ChIR haplotypes for each line, with similar numbers of peaks overall. We found much more complicated patterns from a broiler line from a commercial breeder and a flock of red junglefowl, but trios of parents and offspring from another commercial chicken line show that the complicated patterns are due to heterozygosity, indicating a relatively stable number of peaks within haplotypes of these birds. Some ChIR-AB1 peaks were found in all individuals from the commercial lines, and some of these were shared with red junglefowl and the experimental lines derived originally from egg-laying chickens. Overall, this analysis suggests that there are some simple features underlying the apparent complexity of the ChIR locus.