Fig 1.
The primer design captures full-length HLA class I genes (HLA-A, -B, -C) and exons 2–4 of DRB1 and DQB1 genes. There are two multiplex primer sets: Class I primer mix includes HLA-A, -B, -C, and Class II primer mix includes DRB1 and DQB1.
Fig 2.
High-throughput NGS workflow begins with multiplex long range PCR of A, B, C and DRB1, DQB1. After PCR, amplicons are purified and pooled in equimolar concentrations. Sheared amplicons then undergo library preparation by using the Illumina TruSeq Nano Kit. To maximize throughput, each clinical sample is labeled with unique dual indices. 2×250 bp paired-end sequence data from the Illumina MiSeq are exported and analyzed using Omixon Twin1.0.7, with 3.19.0 IMGT/HLA database serving as the reference.
Fig 3.
Comparison of DNA concentration in buccal swab and blood samples.
The average concentration of buccal-isolated DNA was 3.96 ± 3.74 ng/μL in 100μL volume, which was significantly lower than DNA derived from blood samples (23.99 ± 9.24 ng/μL).
Fig 4.
Long-range PCR products and gel electrophoresis.
a) Gel electrophoresis of Class I (HLA-A, B, C) using a multiplex primer set. b) Gel electrophoresis of Class II (DQB1 and DRB1) using a multiplex primer set. White box denotes PCR failure. P and N refer to positive control and negative control, respectively.
Fig 5.
Coverage data taken from a representative sample.
The sample was aligned using GenDX Version1.9.0. Blue box, UTR; yellow box, exon. black line, intron. Red box shows the low coverage region in intron 3 of all DRB1 alleles. Uniform coverage was achieved in most loci with the exception of coverage bias found in intron 3 of DRB1 alleles due to the (GT)x(GA)y motif.
Table 1.
NGS typing results.
Table 2.
Null allele detection by using NGS in 10,063 buccal swab samples.
Fig 6.
Novel and rare alleles detected by NGS in 10,063 samples.
Sequence data was analyzed using Omixon HLA Twin V1.0.7 (3.19.0 IMGT/HLA database). a) Percentage of SBT-confirmed exon novelties shown by HLA locus. b) Example of a novel allele detected by NGS and confirmed using SBT. SBT was unable to determine the cis-trans phase of the exon novelty; in contrast, parallel sequencing by NGS clearly established the phase and location of the novel variant in the B*40:02:01 new allele. c) Percentage of rare alleles detected shown by HLA locus.
Table 3.
Exon novelty detection by Omixon Twin 1.0.7 through 10,063 buccal swab samples.
Table 4.
Rare alleles detected by NGS that are not present on CWD 2.0.0 in 10,063 samples.