- Email: [email protected]
Full length phased sequencing of HLA class I samples reveals deeper insight in sequence variety
Abstracts / Human Immunology 76 (2015) 38–167
P118
FULL LENGTH PHASED SEQUENCING OF HLA CLASS I SAMPLES REVEALS DEEPER INSIGHT IN SEQUENCE VARIETY. Kathrin Lang a, Ines Wagner a, Bianca Schoene a, Gerhard Schöfl a, Carolin Zweiniger a, Christoph König b, Sylvia Clausing c, Yannick Duport c,d, Nicola Gscheidel c, Andreas Dahl c, Juergen Sauter e, Vinzenz Lange a, Irina Boehme a, Alexander Schmidt a,e. a DKMS Life Science Lab, Dresden, Germany; b Pacific Biosciences, Menlo Park, CA, United States; c CRTD-Centrum for Regenerative Therapies, Dresden, Germany; d Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany ; e DKMS German Bone Marrow Donor Center, Tübingen, Germany. Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated ‘‘high resolution’’ (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2– 4.4 kb. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48–72 amplicons were sequenced full length and phased in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was achieved using the SMRT portal. Sequence analysis was performed using NGSengine software (GenDx). Results: We successfully performed full-length gene sequencing of 1003 samples, harboring ambiguous typings of either HLA-A (n = 46), HLA-B (n = 304) or HLA-C (n = 653). Despite the high per-read raw error rates typical for SMRT sequencing (15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. Unambiguous allelic resolution was achieved for all samples. We observed novel intronic, exonic as well as UTR sequence variations for many of the alleles covered by our data set. This included sequences of 600 individuals with HLA-C⁄07:01/C⁄07:02 genotype revealing the extent of sequence variation outside the exons 2 and 3. Conclusion: Here we present a whole gene amplification and sequencing approach for HLA class I genes. The maturity of this approach was demonstrated by sequencing more than 1000 samples, achieving fully phased allelic sequences. Extensive sequencing of one common allele combination hints at the yet to discover diversity of the HLA system outside the commonly analyzed exons.
P119
EVALUATION OF YIELD AND INTEGRITY OF DNA SAMPLES EXTRACTED FROM CORD BLOOD SEGMENTS AND RED CELLS FRACTIONS USING THE MAXWELLÒ RSC INSTRUMENT. Renata Santos, Angelica DeOliveira, Jennifer Deitz, Porsha Barfield, Amanda Dusenberry, Wendy Hanshew, Dong-Fen Chen. Duke University Medical Center, Durham, NC, United States. Aim: The number of cord blood samples received for a variety of applications has increased in our laboratory. This has led us to evaluate an automated method to improve the TAT and yield of the DNA extraction. Our current procedure for cord blood samples requires an overnight incubation that is labor intensive. Cord blood segments and frozen red cell fractions were received from the Duke Cord Blood Bank to help us evaluate the efficiency and yield of the Maxwell Rapid Sample Concentration (RSC) automation recently released by Promega. Method: The initial volume of the samples received for the study were; 100 ll cord blood segments and 600 ll frozen red cell fractions. The average of the total nucleated cell count was 7.4 107cells/ml for the cord segments and 1.6 107cells/ml for the red cell fractions. The samples were thawed, RNAse and Proteinase K was added, followed by a 30 min incubation. Then each sample was placed into the Maxwell RSC automation in a designated slot. The Maxwell RSC uses a paramagnetic particle to optimize sample capture, washing, and purification by a cellulose-based binding of nucleic acids. The final elution volume was determined to be 100 ll and the equipment Blood Protocol was used for all extractions. The total time for the extraction is 35 min (hands off). HLA typing were performed using; RT-PCR, Luminex, SSP and SBT. Results: Nine samples were isolated by Maxwell RSC (six cord blood and three cell fractions). The DNA yield from the cord segments ranged from 130 ng/ll to 477 ng/ll with the total average yield of 34 lg. The red cells fraction range was from 424 ng/ll to 715 ng/ll with the total average yield of 50 lg. The 260/280 OD ratios were within 1.8–2.0 for both sample sources. Each DNA extraction had its integrity evaluated with low melting agarose gel electrophoresis. The three DNA samples were typed by all of our routine HLA typing methodologies generated successful HLA typing. Conclusion: The Maxwell RSC appears to have high quality results, easy and reliable operation, flexible protocol, and it reduces the TAT for our laboratory to obtain the DNA from cord blood samples requiring HLA typing.
121
P118
FULL LENGTH PHASED SEQUENCING OF HLA CLASS I SAMPLES REVEALS DEEPER INSIGHT IN SEQUENCE VARIETY. Kathrin Lang a, Ines Wagner a, Bianca Schoene a, Gerhard Schöfl a, Carolin Zweiniger a, Christoph König b, Sylvia Clausing c, Yannick Duport c,d, Nicola Gscheidel c, Andreas Dahl c, Juergen Sauter e, Vinzenz Lange a, Irina Boehme a, Alexander Schmidt a,e. a DKMS Life Science Lab, Dresden, Germany; b Pacific Biosciences, Menlo Park, CA, United States; c CRTD-Centrum for Regenerative Therapies, Dresden, Germany; d Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany ; e DKMS German Bone Marrow Donor Center, Tübingen, Germany. Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated ‘‘high resolution’’ (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2– 4.4 kb. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48–72 amplicons were sequenced full length and phased in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was achieved using the SMRT portal. Sequence analysis was performed using NGSengine software (GenDx). Results: We successfully performed full-length gene sequencing of 1003 samples, harboring ambiguous typings of either HLA-A (n = 46), HLA-B (n = 304) or HLA-C (n = 653). Despite the high per-read raw error rates typical for SMRT sequencing (15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. Unambiguous allelic resolution was achieved for all samples. We observed novel intronic, exonic as well as UTR sequence variations for many of the alleles covered by our data set. This included sequences of 600 individuals with HLA-C⁄07:01/C⁄07:02 genotype revealing the extent of sequence variation outside the exons 2 and 3. Conclusion: Here we present a whole gene amplification and sequencing approach for HLA class I genes. The maturity of this approach was demonstrated by sequencing more than 1000 samples, achieving fully phased allelic sequences. Extensive sequencing of one common allele combination hints at the yet to discover diversity of the HLA system outside the commonly analyzed exons.
P119
EVALUATION OF YIELD AND INTEGRITY OF DNA SAMPLES EXTRACTED FROM CORD BLOOD SEGMENTS AND RED CELLS FRACTIONS USING THE MAXWELLÒ RSC INSTRUMENT. Renata Santos, Angelica DeOliveira, Jennifer Deitz, Porsha Barfield, Amanda Dusenberry, Wendy Hanshew, Dong-Fen Chen. Duke University Medical Center, Durham, NC, United States. Aim: The number of cord blood samples received for a variety of applications has increased in our laboratory. This has led us to evaluate an automated method to improve the TAT and yield of the DNA extraction. Our current procedure for cord blood samples requires an overnight incubation that is labor intensive. Cord blood segments and frozen red cell fractions were received from the Duke Cord Blood Bank to help us evaluate the efficiency and yield of the Maxwell Rapid Sample Concentration (RSC) automation recently released by Promega. Method: The initial volume of the samples received for the study were; 100 ll cord blood segments and 600 ll frozen red cell fractions. The average of the total nucleated cell count was 7.4 107cells/ml for the cord segments and 1.6 107cells/ml for the red cell fractions. The samples were thawed, RNAse and Proteinase K was added, followed by a 30 min incubation. Then each sample was placed into the Maxwell RSC automation in a designated slot. The Maxwell RSC uses a paramagnetic particle to optimize sample capture, washing, and purification by a cellulose-based binding of nucleic acids. The final elution volume was determined to be 100 ll and the equipment Blood Protocol was used for all extractions. The total time for the extraction is 35 min (hands off). HLA typing were performed using; RT-PCR, Luminex, SSP and SBT. Results: Nine samples were isolated by Maxwell RSC (six cord blood and three cell fractions). The DNA yield from the cord segments ranged from 130 ng/ll to 477 ng/ll with the total average yield of 34 lg. The red cells fraction range was from 424 ng/ll to 715 ng/ll with the total average yield of 50 lg. The 260/280 OD ratios were within 1.8–2.0 for both sample sources. Each DNA extraction had its integrity evaluated with low melting agarose gel electrophoresis. The three DNA samples were typed by all of our routine HLA typing methodologies generated successful HLA typing. Conclusion: The Maxwell RSC appears to have high quality results, easy and reliable operation, flexible protocol, and it reduces the TAT for our laboratory to obtain the DNA from cord blood samples requiring HLA typing.
121