Publications

One million samples typed by NGS: lessons learned

Lange, V., Boehme, I., Paul, P. et al.

Aim
DKMS Life Science Lab adopted an NGS based HLA typing workflow in early 2013. Meanwhile more than a million samples have been successfully typed by NGS forming the basis for this review of the chances and pitfalls of such an approach.

Methods
Our NGS workflow is based on direct sequencing of PCR amplified exons on Illumina MiSeq instruments. Exons 2 and 3 of HLA-A, B, C, DRB1, DQB1 and DPB1 are amplified by PCR on Fluidigm Access Array chips. Recently the profile was extended to cover in addition ABO, Rhesus and CCR5. Amplicons of up to 768 samples are pooled for a sequencing run. Sequencing data is analysed by the in-house developed software neXtype.

Results
The rather simple workflow proved to be very robust. Loading of the Fluidigm chips is critical and, due to the low volumes, needs careful optimization and regular maintenance of the involved liquid handlers. PCR continuously yielded sufficient product for loading on the sequencers. The MiSeqs in general produce very high quality sequence data. Low diversity samples like amplicon products pose a particular challenge to the MiSeqs but are meanwhile handled well. However, care must be taken to keep the rate of very short PCR artefacts low as those may negatively impact the overall quality. For analysis of the data care must be taken to identify crossover PCR products which may resemble true HLA alleles. In addition, due to PCR artefacts the rate of error at a particular position might be significantly higher than indicated by the q-values. When taking this into account software like neXtype can provide highly automated and accurate results at a fraction of the time of Sanger data analysis. Given adequate software we identified imbalanced amplification as the main source of error. Rigorous quality control of every reagent batch with regard to even amplification is essential. In addition samples with DNA concentrations below 3 ng/μl should not be applied to 192 Fluidigm chips to avoid random loss of alleles.

Conclusions
Switching to NGS as the primary tool for HLA typing was highly beneficial for the lab. The NGS based workflow has proven superior to the previous Sanger based workflow in every aspect: Costs, hands on time, resolution, turnaround time, stability and scalability. We conclude that NGS technology has matured to a point where it is appropriate for everyday routine operation.

Citation

Lange, V., Boehme, I., Paul, P. et al. "One million samples typed by NGS: lessons learned" Human Immunology (2015): 210