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HLA-DPB typing by direct fluorescent sequencing
130
Abstracts
B-7.4 #247
COMPUTERIZED HIGH RESOLUTION SEQUENCE BASED HLA-TYPING. Erik Rozemuller, Leone Versluis, Anne Bouwens, Marcel Tilanus. Diagnostic DNA Laboratory, Academic Hospital Utrecht, The Netherlands. Sequence based typing (SBT) will be one of the major techniques for high resolution HLA-typing. For SBT the sequence of the most polymorphic exon of a locus is used to identify the alleles. Applicability of SBT depends on the determination of quantified sequence data and on computerized sequence data analysis resulting in an allele assignment. We developed software to quantify sequence data and to perform allele assignment automatically. Quantified sequence data are obtained by sequencing the amplified exon using an automated sequencer (ABI, 373A), thus obtaining peak heights for each sequence position. In case of heterozygosity, the determined sequence is a mixture of the sequences of the two involved alleles. Software is developed to identify heterozygous peaks and to assign NC-IUB codes to the corresponding positions. Allele assignment is performed after comparison of the determined sequence with all known allele sequences and their combinations. Alignment at constant positions is used as quality control for the sequence quality. Polymorphic positions which are crucial for the allele assignment are identified and peak heights corresponding to those positions are used as quality control for the allele assignment. Successful DPB typing of 89 samples as performed in a blind study allows the assumption that SBT in which this software analysis is included permits a reliable high resolution HLA typing.
B-7.4 #248
HLA-DPB TYPING BY DIRECT FLUORESCENT SEQUENCING. Leone Verslui~ ~, Erik Rozemuller1, Susan Tonks2, Steven Marsh 2, Anne Bouwens 1, Julia Bodmer2, Marcel Tilanusx. ~Diagnostic DNA Laboratory, Academic Hospital Utrecht the Netherlands. 2Tissue Antigen Laboratory, Imperial Cancer Research Fund London UK. To discriminate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high resolution DPB typing. The most refined DNA typing until now is SSO-typing; new oligonucleotides can be selected to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic hypervariable regions and has the advantage of being independent from known sequence information. We have developed a new DNA based typing system; it is based upon direct sequencing amplified DNA with fluorescent labelled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. Here we describe the DPB typing results of a panel of 89 previously SSO typed DNA samples. Sequencing Based Typing (SBT) was performed in a blind study in Utrecht. 86 samples gave identical SBT and SSO results. The remaining 3 samples could not be typed by the general approach since two different pairs of alleles were found which result in identical heterozygous combinations. This phenomenon can also occur in other allelic couples and can be resolved using group specific sequence primers. We conclude, based upon these results that our SBT for DPB is reliable. Moreover, the method is easy to perform on a routine basis, since both sequencing (373A ABI) and allele assignment are automated. With this method the maximum of information is obtained and the system is easily applicable to other genesystems.
Abstracts
B-7.4 #247
COMPUTERIZED HIGH RESOLUTION SEQUENCE BASED HLA-TYPING. Erik Rozemuller, Leone Versluis, Anne Bouwens, Marcel Tilanus. Diagnostic DNA Laboratory, Academic Hospital Utrecht, The Netherlands. Sequence based typing (SBT) will be one of the major techniques for high resolution HLA-typing. For SBT the sequence of the most polymorphic exon of a locus is used to identify the alleles. Applicability of SBT depends on the determination of quantified sequence data and on computerized sequence data analysis resulting in an allele assignment. We developed software to quantify sequence data and to perform allele assignment automatically. Quantified sequence data are obtained by sequencing the amplified exon using an automated sequencer (ABI, 373A), thus obtaining peak heights for each sequence position. In case of heterozygosity, the determined sequence is a mixture of the sequences of the two involved alleles. Software is developed to identify heterozygous peaks and to assign NC-IUB codes to the corresponding positions. Allele assignment is performed after comparison of the determined sequence with all known allele sequences and their combinations. Alignment at constant positions is used as quality control for the sequence quality. Polymorphic positions which are crucial for the allele assignment are identified and peak heights corresponding to those positions are used as quality control for the allele assignment. Successful DPB typing of 89 samples as performed in a blind study allows the assumption that SBT in which this software analysis is included permits a reliable high resolution HLA typing.
B-7.4 #248
HLA-DPB TYPING BY DIRECT FLUORESCENT SEQUENCING. Leone Verslui~ ~, Erik Rozemuller1, Susan Tonks2, Steven Marsh 2, Anne Bouwens 1, Julia Bodmer2, Marcel Tilanusx. ~Diagnostic DNA Laboratory, Academic Hospital Utrecht the Netherlands. 2Tissue Antigen Laboratory, Imperial Cancer Research Fund London UK. To discriminate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high resolution DPB typing. The most refined DNA typing until now is SSO-typing; new oligonucleotides can be selected to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic hypervariable regions and has the advantage of being independent from known sequence information. We have developed a new DNA based typing system; it is based upon direct sequencing amplified DNA with fluorescent labelled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. Here we describe the DPB typing results of a panel of 89 previously SSO typed DNA samples. Sequencing Based Typing (SBT) was performed in a blind study in Utrecht. 86 samples gave identical SBT and SSO results. The remaining 3 samples could not be typed by the general approach since two different pairs of alleles were found which result in identical heterozygous combinations. This phenomenon can also occur in other allelic couples and can be resolved using group specific sequence primers. We conclude, based upon these results that our SBT for DPB is reliable. Moreover, the method is easy to perform on a routine basis, since both sequencing (373A ABI) and allele assignment are automated. With this method the maximum of information is obtained and the system is easily applicable to other genesystems.