Authors:
J. Pruschke (Tuebingen, DE)
I. Wagner (Dresden, DE)
D. Schefzyk (Tuebingen, DE)
C. Gnahm (Tuebingen, DE)
B. Schoene (Dresden, DE)
K. Lang (Dresden, DE)
J. Hollenbach (San Francisco, US)
P. Norman (Stanford, US)
V. Lange (Dresden, DE)
A. Schmidt (Tuebingen, DE)
J. Hofmann (Tuebingen, DE)
J. Sauter (Tuebingen, DE)
The KIR gene family plays an important role in the immune system and has been reported to impact the outcome of hematopoietic stem cell transplantation. Due to the polymorphism of these genes, allele-level typing results are difficult to achieve. Previously, neXtype delivered absence/presents calls for the KIR genes. Now, the software has been extended to report KIR at the allelic level in genotype list (GL) string format. We employ a short amplicon approach for next generation sequencing to obtain data from exons 3, 4, 5, 7, 8, and 9 with Illumina sequencers. Allele calling is based on the IPD-KIR library. Copy numbers of the allelic variants per exon are determined employing calibrated read counts. For each gene, neXtype computes a score for each possible combination of exon-specific allele calls as potential results. The scoring evaluates the deviation between the observed copy number per exon and the copy number as needed for a specific result. Special attention is needed for gene-bridging exons as the corresponding sequences cannot be assigned to a specific gene a priori. Validation with a set of 93 pre-typed samples revealed a concordance rate of >99%. To further verify consistency, a set of 190 samples (16 loci per sample) was processed 10 times. Out of the 3040 loci, 3025 were typed consistently. 2043 of these results yielded allelic level, 222 loci could be typed as present only, and 760 loci as absent. Of the 15 erroneous results, 13 are due to discrepant copy numbers while two are due to discrepancies on absence/presence level. In our high-throughput workflow, 197,071 samples were assigned a valid KIR typing within 3 months. Of these, 84% were processed automatically. For 1,895,256 loci, allelic level was achieved while 389,881 were reported as present, and 867,999 as absent. In summary, we show that high-throughput KIR allele-level typing is possible with a low error rate. Consequently, high-resolution KIR genotyping is included into DKMS recruitment typing profile.