OR26 KIR typing of 100.000 donor samples by next generation sequencing (NGS)

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• 作者：Vinzenz Lange¹ ; Ines Wagner¹ ; Kathrin Lang¹ ; Patrick Paul¹ ; Johanna M. Andreas¹ ; Philipp Quenzel¹ ; Arend Grosse¹ ; Bianca Schö ; ne¹ ; Lisa Hedrich¹ ; Carmen Schwarzelt¹ ; Daniel M. Baier² ; Jan A. Hofmann² ; Jü ; rgen Sauter² ; Angela Lucaci-Timoce² ; Julia Pingel² ; Irina Bö ; hme¹ ; Alexander H. Schmidt¹ ; ²
• 刊名：Human Immunology
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：76
• 期：supp_S
• 页码：10
• 全文大小：37 K

文摘

Typing of potential donors for all transplantation relevant factors at registration can speed up donor selection which benefits certain patients. Despite several studies regarding the effect of the KIR repertoire on the outcome of hematopoietic stem cell transplantation (HSCT), KIR typing data is commonly not available for donor selection – to some extent due to the costs of conventional KIR typing methods. Our amplicon-based NGS typing approach has reduced HLA typing costs considerably. Here, we report a method for cost effective high-throughput KIR typing that enables the addition of KIR typing to the standard profile for all newly registered donors.

Methods

Exons 4, 5 and 7 are amplified in three PCR reactions using primer mixes targeting all KIR genes. The PCR products contain sample specific identification sequences and can therefore be combined for joint sequencing on Illumina MiSeq or HiSeq instruments. Up to 4800 samples are sequenced on one 2 × 250 rapid run HiSeq flowcell yielding on average about 60,000 reads per sample evenly split between HLA and KIR. Sequencing data is analyzed by neXtype, an inhouse software, and currently yields presence/absence calls for the KIR genes with KIR2DS4 and KIR2DS4N being distinguished and KIR2DL5A and KIR2DL5B being combined into KIR2DL5.

Results

Validation yielded 100% concordance with the pretypings for all 109 samples passing the predefined internal quality criteria. Within the first 4 months in 2015 we performed successful KIR typing for more than 100.000 samples. Initial analysis indicates that haplotypes lacking the KIR core genes 3DP1 and 2DL4 are more common than previously anticipated.

Conclusion

Our amplicon-based NGS typing approach enables us to type up to 5000 samples/day for KIR. Typing costs including KIR increased only moderately compared to the former profile including 6 HLA loci (A, B, C, DRB1, DQB1 and DPB1), CCR5, and blood groups ABO and Rh. This facilitated DKMS to expand the profile for all newly registered donors to include KIR typing.