P095 : HYBRID READS, A QUALITY VALUE FOR HLA AMPLIFICATION: MEASURED WITH NGSengine

Aim Next Generation Sequencing (NGS) technologies are more frequently used to determine HLA genotypes. Since all sequence reads originate from a single molecule, in most cases the nucleotide sequences of the paternal and maternal alleles are determined separately. During PCR amplification the DNA template is amplified, resulting in copies of the original template. However, this process is not perfect, leading to wrong bases in individual molecules. These errors can be identified in NGS, where it is considered noise. In addition, hybrid molecules are generated which represents reactions which start at one molecule, but at some point the product re-anneals to another template, and continues to elongate. Especially if a template originates from a heterozygous sample, and covers multiple positions where the maternal and paternal allele differ, these hybrid molecules can be detected by NGS. In this study we quantified the hybrid molecules as hybrid reads by NGS, and applied it as a quality value for the PCR amplification. Methods HLA gene amplicons were generated with GenDx’ NGSgo-AmpX in independent experiments in different labs. NGS sequencing was done on Illumina MiSeq, Ion Torrent PGM and PacBio RS. NGSengine, a software package to perform HLA typing based on NGS data was used to quantify the number of hybrid reads in several different HLA gene amplification systems. The amount of hybrid reads was compared to other HLA gene amplification strategies. Results Detailed analyses is currently be carried out. We will present the effect of cycling conditions on the generation of hybrid molecules. Furthermore we will show to what extend this phenomenon is related to one or more platforms. We will come up with measures such that this may be exploited as a quality parameter for NGS HLA Typing. Conclusions NGSengine allows for easy investigation of hybrid reads. Hybrid reads can be used as quality control for amplification robustness. R. Kooter: Employee; Company/Organization; GenDx. M.T. Penning: Employee; Company/Organization; GenDx. W. Mulder: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx.