2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

BMC Genomics

Research Article

Gerhard Schöfl¹  Philipp Quenzel¹  Vinzenz Lange¹  Kathrin Lang¹  Irina Böhme¹  Alexander H. Schmidt²  Julia Pingel³  Jan A. Hofmann³  Jürgen Sauter³ 

[1] DKMS Life Science Lab, Blasewitzerstr. 43, 01307, Dresden, Germany;DKMS Life Science Lab, Blasewitzerstr. 43, 01307, Dresden, Germany;DKMS, Kressbach 1, 72072, Tübingen, Germany;DKMS, Kressbach 1, 72072, Tübingen, Germany;

关键词: Next generation sequencing;    HLA genotyping;    High resolution;    High throughput;    Amplicon PCR;    DKMS;    Primer dimers;    PCR chimerism;    Novel alleles;   

DOI  :  10.1186/s12864-017-3575-z

 received in 2016-12-13, accepted in 2017-02-09,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAt the DKMS Life Science Lab, Next Generation Sequencing (NGS) has been used for ultra-high-volume high-resolution genotyping of HLA loci for the last three and a half years. Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments.ResultsBetween January 2013 and June 2016, 2,714,110 samples largely from German, Polish and UK-based potential stem cell donors have been processed. 98.9% of all alleles for the targeted HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) were typed at high resolution or better. Initially a simple three-step workflow based on nanofluidic chips in conjunction with 4-primer amplicon tagging was used. Over time, we found that this setup results in PCR artefacts such as primer dimers and PCR-mediated recombination, which may necessitate repeat typing. Split workflows for low- and high-DNA-concentration samples helped alleviate these problems and reduced average per-locus repeat rates from 3.1 to 1.3%. Further optimisations of the workflow included the use of phosphorothioate oligos to reduce primer degradation and primer dimer formation, and employing statistical models to predict read yield from initial template DNA concentration to avoid intermediate quantification of PCR products. Finally, despite the populations typed at DKMS Life Science Lab being relatively homogenous genetically, an analysis of 1.4 million donors processed between January 2015 and May 2016 led to the discovery of 1,919 distinct novel HLA alleles.ConclusionsAmplicon-based NGS HLA genotyping workflows have become the workhorse in high-volume tissue typing of registry donors. The optimisation of workflow practices over multiple years has led to insights and solutions that improve the efficiency and robustness of short amplicon based genotyping workflows.

【 授权许可】

CC BY   
© The Author(s). 2017