Wietse Mulder

158-P: NGSengine EARLY ACCESS PROGRAM: FINDINGS IN A MULTICENTER EVALUATION

Aim NGS technology is developing fast and has come to a point where it can be applied routinely. Many HLA typing laboratories are considering introducing NGS technologies in their diagnostics routines. To facilitate HLA typing by NGS, we developed NGS-g o ®, a generic HLA gene amplification strategy, and NGSengine®, a software package to perform HLA typing based on NGS data. Methods An NGS Early Access Program (EAP) was set up to determine if our NGS solutions fulfill the needs and requirements of users within the HLA field. This EAP gives laboratories the chance to evaluate both NGS-g o ® and NGSengine® with their NGS instrument of choice, and even more important, it gives laboratories influence on the design of the software package.In the NGS EAP over 20 laboratories from various geographical regions participated. All labs were given access to NGS-go® reagents and NGSengine® software and were stimulated to provide feedback on the performance. Based on their feedback, NGSengine® was adjusted and significantly improved. During the EAP new updates of NGSengine® are monthly released. Results Based on feedback received, the profiles of the participating labs were determined. Here, we present the NGS experience feedback received from the participating labs. We will also present statistics on the equipment used in the participating labs and show the laboratory profiles of the labs using NGS. Conclusions • 5 different platforms were evaluated by more than 20 participating labs located world-wide • Participating labs had very variable levels of NGS experience • Participating labs possess different favorite NGS platforms • Many of the participating labs are in the early days of HLA testing with NGS and have not yet established a workflow • Valuable feedback was obtained from participants for furtherimprovement of NGSengine®. Penning: GenDx: Employee. Kooter: GenDx: Employee. Ruzius: GenDx: Employee. Mulder: GenDx: Employee; Stockholder. Rozemuller: GenDx: Employee; Stockholder.

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.

P077 : IMPROVED SBTENGINE BATCH ANALYSIS MODULE

Aim HLA typing by Sanger Sequencing is a high-resolution typing method which depend on the correct analysis of the sequence traces. Using a combination of Core sequencing and Group Specific Sequencing reactions (GSSPs) results in allele level typing in the vast majority of samples. However, the reliability of the results depend on the sequence quality and the accuracy of the manual inspection and editing. SBTengine® is a software package which optimizes the SBT analysis workflow. CWD lists and genotype expected frequencies are applied to focus on the crucial steps of the analysis. GSSP advisor and GPS predictor are tools used to minimize the amount of work needed to come to the required resolution. However, an accurate manual inspection and editing are still required. Methods To reduce the manual inspection and editing work, we developed a module for batch analysis, which analyzes Sanger SBT data to come to an HLA typing result. The analysis includes an extensive evaluation of the sequence quality at every position in the context of the other traces of a sample. This means that the results are based on the combined quality of all traces. The module also optimizes the result in the context of the expected frequencies of the genotyping results. The quality of the traces directly influences the possibilities of batch analysis, which is a balance between acceptance of some data which turns out to result in inaccuracy of determining the typing results, or rejection, which results in rejection of a sample for automated typing. Result Here we will present an update on the batch analysis feature. The results of the batch analysis depend on the basic quality of the traces. With good quality data, in the majority of cases the correct genotype is identified, which makes the module applicable for registry typing. A small percentage of samples may not come to the correct genotype, although it is expected that this concerns only minor subtypes. For donor-recipient typing, the module can perform the first analysis, which always has to be followed by an intensive evaluation by the lab supervisor. Conclusion In conclusion: The batch analysis leads to a reduction of editing, and offers a method for increased efficiency of Sanger SBT data analysis. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. J. Geerligs: Employee; Company/Organization; GenDx. M.T. Penning: Employee; Company/Organization; GenDx. W. Mulder: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx.

163-P High resolution HLA typing for companion diagnostics using the next generation DNA sequencing strategy: Pyrosequencing

Recently, requests for accurate HLA typing have been significantly raised in the area of prevention and personalized health care. Evidence has been presented by several groups worldwide that HLA genes, and in particular some HLA alleles, are associated with diseases or alternatively with adverse effects of certain drugs. For instance Celiac Disease is strongly associated with HLA-DQ2 and HLA-DQ8, as both underlying HLA-DQA1 / HLA-DQB1 genotypes predispose for disease development. Another example is that HIV positive patients carrying the HLA-B*57:01 allele should not be treated with Abacavir, since these patients develop hypersensitivity to this drug. On the other hand, it has also been shown that certain alleles within the groups of HLA-B*27 and HLA-B*57 slow down the development and progress of AIDS in HIV positive patients, as compared to patients without this allele.