- Email: [email protected]
158-P Complete characterization of 12 MHC genes for multiple samples by next-generation sequencing
S120
Abstracts
158-P
COMPLETE CHARACTERIZATION OF 12 MHC GENES FOR MULTIPLE SAMPLES BY NEXTGENERATION SEQUENCING. Deborah Ferriola,1 Curt Lind,1 Steven Heron,1 Anh Huynh,1 Larissa Slavich,1 Ariella Sasson,2 Xiaowu Gai,2 Dimitri Monos.1,3 1Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, USA; 2Bioinformatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, USA; 3Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA. Aim: Determine whether the high throughput of Next-Generation Sequencing (NGS) would allow complete genomic sequencing of 12 MHC genes for multiple samples in a single run. Methods: Three samples from homozygous cell lines (RML, MT14B and QBL) were obtained from the IHWG. Twelve PCR reactions were performed for each sample amplifying 12 complete genes (HLA-A, B, C, E, G, DRB1, DQB1, DQA1, DPB1, DRB3/4/5, MICA and MICB) ranging in length from 3kb - 15kb and spanning ⬃93kb total. Following amplification, samples were prepared for shotgun sequencing on a GS FLX (Roche) using the Rapid Library Prep method. Libraries from each gene were tagged with unique identifiers and were pooled for sequencing both independently and as a mixture of 2 samples to determine whether the method could correctly characterize the known alleles in heterozygosity. Each pool was sequenced on 1/8th of a picotiter plate. Sequence data were analyzed with software analysis programs: Assign-NG (Conexio Genomics), HLACaller (Broad Institute) and Sequencher (Gene Codes). Results: More than 75,000 sequence reads were generated for each sample with each locus having 1,300 to 13,000 reads with a median read length of 278 bases. Each gene was characterized at all bases with an average read depth of ⬃250. Unambiguous typing was obtained for all loci and for all samples both as homozygotes and when mixed to simulate heterozygosity. Analyses of previously sequenced regions were concordant with the known sequence. Previously unknown typing was obtained for RML (HLA-E and HLAG) and MT14B (HLA-G, MICA and MICB). Furthermore full gene sequence was obtained for several alleles (A*02:04, DRB1*04:04:01, DRB1*16:02:01, MICA*009:01, MICB*005:04) that were previously only partially characterized, in addition to a new HLA-G allele. Conclusions: This experiment demonstrates the great potential of this technology as a research and diagnostic tool for the complete and unambiguous typing of multiple samples at multiple genomic regions in parallel.
159-P
HISTOCOMPATIBILITY TESTING WITH DNA ISOLATED FROM BUCCAL SWABS USING THE Maxwell® 16 BUCCAL SWAB LEV DNA PURIFICATION KIT. Rebecca Gorshe,1 Jennifer Schiller,2 Thomas Ellis,2 Tracy Fisher,2 Trista Schagat.1 1Scientific Applications Support, Promega Corporation, Madison, WI, USA; 2Histocompatibility and Immunogenetics, Blood Center of Wisconsin, Milwaukee, WI, USA. Aim: Histocompatibility testing to support organ and stem cell transplantation involves Human Leukocyte Antigen (HLA) typing of major histocompatibility complex (MHC) genes. Typically, DNA is extracted and analyzed by Sequence Specific Oligonucleotides (SSO) typing, Sequence Specific Priming (SSP) typing, Sequence Based Typing (SBT), or Short Tandem Repeat (STR) analysis. DNA isolation from buccal swabs is an advantageous alternative to a blood draw. Here we describe using the Maxwell® 16 Buccal Swab LEV DNA Kit for the automated extraction of DNA from buccal swabs for use in a variety of histocompatibility testing assays and qPCR. Methods: Buccal cell samples were collected using cytobrushes and cotton swabs. DNA was isolated using the Maxwell® 16 System with the Maxwell® 16 Buccal Swab LEV DNA Kit. Overall yield, concentration and purity were assessed. DRB5 SSP analysis was performed with the DRB5 SSP Unitray® System. rSSO and sequence-based typings were performed for HLA-A, -B, -C, -DRB1, -DQB1, and –DPB1. HLA haplotyping was assessed by STR analysis. qPCR analysis was performed on a subset to test for inhibitors. Results: DNA concentrations were 53ng/l to 256ng/l, with a mean concentration of 127ng/l. DNA purities were A260/A280 1.73 to 1.99, with a mean of 1.87. Samples tested in the SSP assay correlated with previous typing. Samples tested in downstream assays for HLA SSO typing, and HLA haplotyping by STR were successful. qPCR confirmed no detectable PCR inhibitors. Conclusions: We have demonstrated use of the Maxwell® 16 Buccal Swab LEV DNA Kit for automated DNA extraction from buccal swabs. The DNA extracted was compatible with qPCR and SSO/SSP/SBT/STR analyses. Use of this automated purifcation system allowed the user consistent recovery of DNA from the samples that was sufficient for accurate allele determination. In addition, DNA was isolated in about an hour with minimal hands on time, allowing for quick turnaround when time may be critical for HLA typing. Gorshe: Promega Corporation: Employee. Schagat: Promega Corporation: Employee.
Abstracts
158-P
COMPLETE CHARACTERIZATION OF 12 MHC GENES FOR MULTIPLE SAMPLES BY NEXTGENERATION SEQUENCING. Deborah Ferriola,1 Curt Lind,1 Steven Heron,1 Anh Huynh,1 Larissa Slavich,1 Ariella Sasson,2 Xiaowu Gai,2 Dimitri Monos.1,3 1Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, USA; 2Bioinformatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, USA; 3Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA. Aim: Determine whether the high throughput of Next-Generation Sequencing (NGS) would allow complete genomic sequencing of 12 MHC genes for multiple samples in a single run. Methods: Three samples from homozygous cell lines (RML, MT14B and QBL) were obtained from the IHWG. Twelve PCR reactions were performed for each sample amplifying 12 complete genes (HLA-A, B, C, E, G, DRB1, DQB1, DQA1, DPB1, DRB3/4/5, MICA and MICB) ranging in length from 3kb - 15kb and spanning ⬃93kb total. Following amplification, samples were prepared for shotgun sequencing on a GS FLX (Roche) using the Rapid Library Prep method. Libraries from each gene were tagged with unique identifiers and were pooled for sequencing both independently and as a mixture of 2 samples to determine whether the method could correctly characterize the known alleles in heterozygosity. Each pool was sequenced on 1/8th of a picotiter plate. Sequence data were analyzed with software analysis programs: Assign-NG (Conexio Genomics), HLACaller (Broad Institute) and Sequencher (Gene Codes). Results: More than 75,000 sequence reads were generated for each sample with each locus having 1,300 to 13,000 reads with a median read length of 278 bases. Each gene was characterized at all bases with an average read depth of ⬃250. Unambiguous typing was obtained for all loci and for all samples both as homozygotes and when mixed to simulate heterozygosity. Analyses of previously sequenced regions were concordant with the known sequence. Previously unknown typing was obtained for RML (HLA-E and HLAG) and MT14B (HLA-G, MICA and MICB). Furthermore full gene sequence was obtained for several alleles (A*02:04, DRB1*04:04:01, DRB1*16:02:01, MICA*009:01, MICB*005:04) that were previously only partially characterized, in addition to a new HLA-G allele. Conclusions: This experiment demonstrates the great potential of this technology as a research and diagnostic tool for the complete and unambiguous typing of multiple samples at multiple genomic regions in parallel.
159-P
HISTOCOMPATIBILITY TESTING WITH DNA ISOLATED FROM BUCCAL SWABS USING THE Maxwell® 16 BUCCAL SWAB LEV DNA PURIFICATION KIT. Rebecca Gorshe,1 Jennifer Schiller,2 Thomas Ellis,2 Tracy Fisher,2 Trista Schagat.1 1Scientific Applications Support, Promega Corporation, Madison, WI, USA; 2Histocompatibility and Immunogenetics, Blood Center of Wisconsin, Milwaukee, WI, USA. Aim: Histocompatibility testing to support organ and stem cell transplantation involves Human Leukocyte Antigen (HLA) typing of major histocompatibility complex (MHC) genes. Typically, DNA is extracted and analyzed by Sequence Specific Oligonucleotides (SSO) typing, Sequence Specific Priming (SSP) typing, Sequence Based Typing (SBT), or Short Tandem Repeat (STR) analysis. DNA isolation from buccal swabs is an advantageous alternative to a blood draw. Here we describe using the Maxwell® 16 Buccal Swab LEV DNA Kit for the automated extraction of DNA from buccal swabs for use in a variety of histocompatibility testing assays and qPCR. Methods: Buccal cell samples were collected using cytobrushes and cotton swabs. DNA was isolated using the Maxwell® 16 System with the Maxwell® 16 Buccal Swab LEV DNA Kit. Overall yield, concentration and purity were assessed. DRB5 SSP analysis was performed with the DRB5 SSP Unitray® System. rSSO and sequence-based typings were performed for HLA-A, -B, -C, -DRB1, -DQB1, and –DPB1. HLA haplotyping was assessed by STR analysis. qPCR analysis was performed on a subset to test for inhibitors. Results: DNA concentrations were 53ng/l to 256ng/l, with a mean concentration of 127ng/l. DNA purities were A260/A280 1.73 to 1.99, with a mean of 1.87. Samples tested in the SSP assay correlated with previous typing. Samples tested in downstream assays for HLA SSO typing, and HLA haplotyping by STR were successful. qPCR confirmed no detectable PCR inhibitors. Conclusions: We have demonstrated use of the Maxwell® 16 Buccal Swab LEV DNA Kit for automated DNA extraction from buccal swabs. The DNA extracted was compatible with qPCR and SSO/SSP/SBT/STR analyses. Use of this automated purifcation system allowed the user consistent recovery of DNA from the samples that was sufficient for accurate allele determination. In addition, DNA was isolated in about an hour with minimal hands on time, allowing for quick turnaround when time may be critical for HLA typing. Gorshe: Promega Corporation: Employee. Schagat: Promega Corporation: Employee.