Suretyper software

Whole genomic DNA was isolated from blood using QIAamp DNA Blood Mini Kit (Qiagen Iberia, Madrid, Spain). Intermediate HLA genotyping resolution (four digits, the corresponding “P” for every 2^nd field allele typing was discarded to clarify the text) was performed with 2μg of genomic DNA using LinkSēq™ HLA ABCDRDQB1 384 kit (One Lambda, Thermo Fisher Scientific España, Madrid, Spain), which allows testing human class I classical HLA-A, -B, -C alleles and class II–DR, -DQ and–DP alleles, including Bw4 and Bw6 genotypes. qPCR was performed using QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), according to manufacturer’s instructions and using the following conditions: 95°C, 2 min; 36 cycles: 95°, 15 seconds; 64°C, 1 min. A dissociation profile was added using the following conditions: 95°C, 15 seconds; 65°C, 30 seconds. The dissociation data corresponding to the melt curve were collected per well between 65°C and 95°C using QuantStudio™ Design & Analysis Software (Thermo Fisher Scientific). These data were exported to SureTyper software (One Lambda) for the interpretation and identification of HLA alleles.

Genomic DNA was extracted as described above. Killer cell Ig-like receptors (KIR) genotyping was performed with 360 ng of genomic DNA using LinkSeq KIR Typing kit (One Lambda, Thermo Fisher Scientific), which allows testing all 15 human KIR genes and 2 pseudogenes, including both full length and deleted forms of 3DP1 and 2DS4 and allele-specific variants in 3DL1, 2DL1. qPCR was performed using StepOne Real-Time PCR System (Thermofisher Scientific) according to manufacturer’s instructions. These data were exported to SureTyper software (One Lambda) for interpretation and identification of KIR genotypes. KIR haplotypes AA and BX were assigned to each patient sample as previously shown (25 ). B haplotypes are defined as those carrying one or more of the following genes: KIR2DL2, -2DL5, -3DS1, -2DS1, -2DS2, -2DS3 and -2DS5; whereas A haplotypes are defined as those that do not carry any of these genes.

KIR genotyping was performed with 360 ng of genomic DNA using LinkSeq KIR Typing kit (One Lambda, Thermo Fisher Scientific). This kit allows testing all 15 human KIR genes and 2 pseudogenes, including both full length and deleted forms of 3DP1 and 2DS4 and allele-specific variants in 3DL1, 2DL1. qPCR was performed using StepOne Real-Time PCR System (Thermo Fisher Scientific) according to manufacturer’s instructions and using the following conditions: 95°C, 2 min; 36 cycles: 95°, 15 seconds; 64°C, 1 min. A dissociation profile was added (95°C, 15 seconds; 65°C, 30 seconds) and dissociation data corresponding to the melt curve of the amplification were collected per well between 65°C and 95°C using StepOne Plus software v2.3. These data were exported to SureTyper software (One Lambda) for interpretation and identification of KIR genotypes.