- Email: [email protected]
190-P: High-resolution KIR genotyping on pooled samples by next generation sequencing
Abstracts
190-P
S107
HIGH-RESOLUTION KIR GENOTYPING ON POOLED SAMPLES BY NEXT GENERATION SEQUENCING. Martha B. Ladner,1 Gordon Bentley,2 Damian Goodridge,3 Henry A. Erlich,2 Elizabeth Trachtenberg.1 1CHORI, Oakland, CA, USA; 2Department of Human Genetics, Roche Molecular Systems, Inc., Pleasanton, CA, USA; 3Conexio Genomics, Australia. Aim: The Killer Cell Immunoglobulin-like Receptor (KIR) gene complex contains a variable number (7-17) of highly polymorphic genes that code for either stimulatory or inhibitory cell surface receptors that act to guide the innate immune response. Evidence suggests that KIR genes are associated with reproductive fitness and many diseases, including autoimmune diseases and survival outcome in hematopoietic cell transplant. To more fully understand the role of the complex in health and disease, we are developing a high-throughput KIR gene sequencing method, taking advantage of the new clonal sequencing technologies which support long reads and large numbers of sequence reads, or depth. Methods: We are using the 454 GS FLX sequencing platform to develop high-throughput sequencing to analyze all KIR genes concurrently in one sequencing run. Using an amplicon approach and pooling 8 DNA samples previously sequenced by the National Marrow Donor Program, we are analyzing the first runs using a software program (Conexio Genomics) to analyze KIR genes at an allelic level. Results: Our preliminary study of KIR genotyping on 8 samples (previously sequenced using Sanger sequencing and cloning techniques) with the Roche FLX 454 returned over 371,000 reads, and yielded an almost complete KIR sequence on all loci and a concordance rate of ⬎98% with known KIR sequences. Further studies are in process to increase the number of samples and increase the depth of reads for all loci. Conclusions: Our preliminary results indicate that this next-generation, clonal sequencing method will work as an alternative, high-throughput method to more traditional methods of KIR sequencing. Bentley: Roche: Employee. Goodridge: Conexio Genomics: Employee. Erlich: Roche: Employee.
191-P
PHOSPHO-FLOW CYTOMETRY: A NOVEL METHOD TO MEASURE ANTIBODY INDUCED ACTIVATION IN ENDOTHELIAL CELLS. Fang Li, Jennifer Chou, Elaine F. Reed. Immunogenetics, University of California Los Angeles, Los Angeles, CA, USA. Aim: Ligation of class I molecules with anti-HLA Ab initiates two signaling outcomes survival vs proliferation depending upon the Ab concentration. Traditionally, signal transduction was measured using Western blot analysis. However, this method is cumbersome and not amenable to studying large numbers of clinical samples. To overcome this problem we developed a novel assay to measure intracellular signal transduction by flow cytomerty. We report for the first time on the application of phospho-flow cytometry to quantifying class I mediated signal transduction in endothelial cells (EC). Methods: Purified IgG from sera developing post-transplant anti-HLA Ab were tested for their capacity to transduce signals in human EC by detecting phosphorylation of S6 ribosomal protein (S6RP), Akt, and ERK. Parallel experiments were performed by Western blot. Results: Fundamental steps in IgG purification, permeabilizing, fixation, cell staining, and cell washing were characterized for accuracy, reproducibility, cell recovery and phosphorylation detection. Treatment of EC with the IgG fraction of anti-HLA Ab positive sera increased the phosphorylation of Akt, S6RP and ERK. Overall performance of the phosphoflow technique was comparable to that obtained by Western blot. Consistent with previous data, low levels of anti-class I Ab (0.1 g/ml) stimulated phoshorylation of Akt and ERK. Treatment of EC with INF-␥ upregulated class II expression and was accompanied by the increased ERK phosphorylation following ligation with anti-class II IgG. Conclusions: Phosphoflow cytometry is a simple, high throughput and quantitative assay to measure phosphorylation at the single cell level and may be useful for translational research and diagnostic testing.
190-P
S107
HIGH-RESOLUTION KIR GENOTYPING ON POOLED SAMPLES BY NEXT GENERATION SEQUENCING. Martha B. Ladner,1 Gordon Bentley,2 Damian Goodridge,3 Henry A. Erlich,2 Elizabeth Trachtenberg.1 1CHORI, Oakland, CA, USA; 2Department of Human Genetics, Roche Molecular Systems, Inc., Pleasanton, CA, USA; 3Conexio Genomics, Australia. Aim: The Killer Cell Immunoglobulin-like Receptor (KIR) gene complex contains a variable number (7-17) of highly polymorphic genes that code for either stimulatory or inhibitory cell surface receptors that act to guide the innate immune response. Evidence suggests that KIR genes are associated with reproductive fitness and many diseases, including autoimmune diseases and survival outcome in hematopoietic cell transplant. To more fully understand the role of the complex in health and disease, we are developing a high-throughput KIR gene sequencing method, taking advantage of the new clonal sequencing technologies which support long reads and large numbers of sequence reads, or depth. Methods: We are using the 454 GS FLX sequencing platform to develop high-throughput sequencing to analyze all KIR genes concurrently in one sequencing run. Using an amplicon approach and pooling 8 DNA samples previously sequenced by the National Marrow Donor Program, we are analyzing the first runs using a software program (Conexio Genomics) to analyze KIR genes at an allelic level. Results: Our preliminary study of KIR genotyping on 8 samples (previously sequenced using Sanger sequencing and cloning techniques) with the Roche FLX 454 returned over 371,000 reads, and yielded an almost complete KIR sequence on all loci and a concordance rate of ⬎98% with known KIR sequences. Further studies are in process to increase the number of samples and increase the depth of reads for all loci. Conclusions: Our preliminary results indicate that this next-generation, clonal sequencing method will work as an alternative, high-throughput method to more traditional methods of KIR sequencing. Bentley: Roche: Employee. Goodridge: Conexio Genomics: Employee. Erlich: Roche: Employee.
191-P
PHOSPHO-FLOW CYTOMETRY: A NOVEL METHOD TO MEASURE ANTIBODY INDUCED ACTIVATION IN ENDOTHELIAL CELLS. Fang Li, Jennifer Chou, Elaine F. Reed. Immunogenetics, University of California Los Angeles, Los Angeles, CA, USA. Aim: Ligation of class I molecules with anti-HLA Ab initiates two signaling outcomes survival vs proliferation depending upon the Ab concentration. Traditionally, signal transduction was measured using Western blot analysis. However, this method is cumbersome and not amenable to studying large numbers of clinical samples. To overcome this problem we developed a novel assay to measure intracellular signal transduction by flow cytomerty. We report for the first time on the application of phospho-flow cytometry to quantifying class I mediated signal transduction in endothelial cells (EC). Methods: Purified IgG from sera developing post-transplant anti-HLA Ab were tested for their capacity to transduce signals in human EC by detecting phosphorylation of S6 ribosomal protein (S6RP), Akt, and ERK. Parallel experiments were performed by Western blot. Results: Fundamental steps in IgG purification, permeabilizing, fixation, cell staining, and cell washing were characterized for accuracy, reproducibility, cell recovery and phosphorylation detection. Treatment of EC with the IgG fraction of anti-HLA Ab positive sera increased the phosphorylation of Akt, S6RP and ERK. Overall performance of the phosphoflow technique was comparable to that obtained by Western blot. Consistent with previous data, low levels of anti-class I Ab (0.1 g/ml) stimulated phoshorylation of Akt and ERK. Treatment of EC with INF-␥ upregulated class II expression and was accompanied by the increased ERK phosphorylation following ligation with anti-class II IgG. Conclusions: Phosphoflow cytometry is a simple, high throughput and quantitative assay to measure phosphorylation at the single cell level and may be useful for translational research and diagnostic testing.