High-resolution, high-throughput HLA genotyping by next-generation sequencing.
High-resolution, high-throughput HLA genotyping by next-generation sequencing.
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DOI：
10.1111/j.1399-0039.2009.01345.x
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发表时间：
2009-11
期刊：
影响因子：
--
通讯作者：
Erlich HA
中科院分区：
文献类型：
--
作者：
Bentley G;Higuchi R;Hoglund B;Goodridge D;Sayer D;Trachtenberg EA;Erlich HA
人类白细胞抗原（HLA）I类和II类基因座是人类基因组中最具多态性的基因。造血干细胞移植需要在多个位点进行等位基因水平的HLA分型，为受体患者选择最匹配的无关供体。在目前用于HLA分型的方法中，杂合子的两个等位基因同时扩增和分型或测序，通常使得难以明确地确定两个等位基因的序列。下一代测序方法并行克隆繁殖数百万个单个DNA分子，然后也并行测序。最近，通过一种这样的下一代测序方法（454 Life Sciences，Inc.）增加到250个核苷酸。这些克隆读段长度使得可以设定外显子内连锁多态性的相位，从而明确确定每个HLA等位基因的序列。在这里，我们证明了这种能力，以及表明，该系统的吞吐量是足够高的，使一个完整的，7个位点的HLA I类和II类分型的24或48个单独的DNA在一个单一的GS FLX测序运行。通过在引物中使用样本特异性内部序列标签（多重识别标签或MID）促进高度多重扩增子测序，该引物允许合并样本，但仍保持将序列分配给特定个体的能力。我们已经整合了Conexio Genomics（Freemantle，Australia）开发的HLA分型软件应用程序，该软件应用程序从454个序列数据中为这7个基因座（HLA-A、-B、-C、DRB 1、DQA 1、DQB 1、DPB 1）以及DRB 3、DRB 4和DRB 5分配HLA基因型。这种HLA测序系统分析嵌合体混合物的潜力在这里通过检测两个纯合细胞系（1/100）的混合物中的罕见HLA-B等位基因，以及通过检测严重联合免疫缺陷病综合征（SCIDS）患者血液中存在的罕见非传播的母体等位基因来证明。
The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.
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影响因子：
20.3
作者：
Kawase, Takakazu;Morishima, Yasuo;Sasazuki, Takehiko
通讯作者：
Sasazuki, Takehiko
影响因子：
158.5
作者：
Petersdorf, EW;Hansen, JA;Anasetti, C
通讯作者：
Anasetti, C
影响因子：
3.2
作者：
Petersdorf, EW
通讯作者：
Petersdorf, EW
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：
Parameswaran P;Jalili R;Tao L;Shokralla S;Gharizadeh B;Ronaghi M;Fire AZ
通讯作者：
Fire AZ