A multi-site study using high-resolution HLA genotyping by next generation sequencing.
A multi-site study using high-resolution HLA genotyping by next generation sequencing.
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DOI：
10.1111/j.1399-0039.2010.01606.x
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发表时间：
2011-03
期刊：
影响因子：
--
通讯作者：
Erlich HA
中科院分区：
文献类型：
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作者：
Holcomb CL;Höglund B;Anderson MW;Blake LA;Böhme I;Egholm M;Ferriola D;Gabriel C;Gelber SE;Goodridge D;Hawbecker S;Klein R;Ladner M;Lind C;Monos D;Pando MJ;Pröll J;Sayer DC;Schmitz-Agheguian G;Simen BB;Thiele B;Trachtenberg EA;Tyan DB;Wassmuth R;White S;Erlich HA
人类白细胞抗原I类和II类基因座的高度多态使得高分辨率的人类白细胞抗原分型具有挑战性。目前的分型方法，包括Sanger测序，由于不完全的基因组覆盖和不能确定HLA单倍型的确定阶段，产生了模棱两可的分型结果。454 Life Sciences GS FLX下一代测序系统与Conexio ATF软件相结合，可以提供非常高分辨率的人类白细胞抗原基因分型。高通量基因分型可以通过使用带有多重识别符(MID)标签的引物来实现，以允许在测序之前汇集来自不同个体的扩增片段。我们进行了一项双盲研究，其中8个实验室使用GS Flx标准化学方法进行了扩增子测序，并在一次测序中对相同的20个样本进行了HLAA-A、-B、-C、DPB1、DQA1、DQB1、DRB1和DRB3、DRB4和DRB5(DRB3/4/5)的基因分型。平均序列阅读长度为250个碱基对(BP)，每个扩增子的平均序列阅读次数为672个，这为等位基因分配提供了信心。在所考虑的1280个基因分型中，95%的病例可以进行基因分型。未能指定基因类型是研究人员程序错误或新等位基因存在的结果，而不是测序技术的失败。在可能分配的情况下，8个位点与已知基因型的符合率在95.3%到99.4%之间，总体符合率为97.2%。我们的结论是，使用GS Flx平台和Conexio ATF软件的克隆性焦磷酸测序可以在高分辨率下可靠地鉴定人类白细胞抗原基因型别。
The high degree of polymorphism at HLA class I and class II loci makes high resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results due to incomplete genomic coverage and inability to set phase for HLA haplotype determination. The 454 Life Sciences GS FLX next generation sequencing system coupled with Conexio ATF software can provide very high resolution HLA genotyping. High throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, and DRB3, DRB4 and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs (bp) and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and Conexio ATF software allows reliable identification of HLA genotypes at high resolution.
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影响因子：
--
作者：
Bentley G;Higuchi R;Hoglund B;Goodridge D;Sayer D;Trachtenberg EA;Erlich HA
通讯作者：
Erlich HA
影响因子：
20.3
作者：
Kawase, Takakazu;Morishima, Yasuo;Sasazuki, Takehiko
通讯作者：
Sasazuki, Takehiko
DOI：
10.3324/haematol.10899
发表时间：
2007-05-01
期刊：
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
影响因子：
--
作者：
Maury, Sebastien;Balere-Appert, Marie-Lorraine;Socie, Gerard
通讯作者：
Socie, Gerard
影响因子：
14.9
作者：
Robinson J;Waller MJ;Fail SC;McWilliam H;Lopez R;Parham P;Marsh SG
通讯作者：
Marsh SG
影响因子：
4.8
作者：
Greinix, HT;Faé, I;Fischer, GF
通讯作者：
Fischer, GF