کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6116646 | 1216358 | 2015 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing
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کلمات کلیدی
Genomic PCRHLA genotypingHSCTMIDVHRNGSCWDRMSSSOPTPGPCRSSPsequence-specific oligonucleotide - oligonucleotide اختصاصی دنبالهSequence-specific primer - آغازگر مخصوصHuman leukocyte antigen - آنتی ژن لوسکسی انسانHLA - آنتیژن گلبول سفید انسانیNext generation sequencing - توالی نسل بعدیDNA sequencing - توالی یابی DNA، تعیین توالی دیانایMismatches - ناسازگاریVery high resolution - وضوح بسیار بالاHematopoietic stem cell transplantation - پیوند مغز استخوانhigh resolution - کیفیت بالا
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ایمونولوژی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array⢠is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Human Immunology - Volume 76, Issue 12, December 2015, Pages 910-916
Journal: Human Immunology - Volume 76, Issue 12, December 2015, Pages 910-916
نویسندگان
F. Yamamoto, B. Höglund, M. Fernandez-Vina, D. Tyan, M. Rastrou, T. Williams, P. Moonsamy, D. Goodridge, M. Anderson, H.A. Erlich, C.L. Holcomb,