VALIDATION OF A NEXT GENERATION SEQUENCING BASEDHLA TYPING SYSTEM
Jonathan Downing, Rebecca Lopes, Aous Al-Ibousi, Heather Dunckley
New Zealand Blood Service National Tissue Typing Laboratory

ABSTRACTMETHODSRESULTS

We have validated and implemented the Illumina TruSight
HLAv2 kit for typing of HLA-A, -B, -C, -DRB, -DQ and -DP for
solid organ and haematopoietic stem cell transplantation. In
addition we have validated the use of this kit on the Eppendorf
epMotion® 5075 liquid handling robot.

The Illumina TruSight HLAv2 kit was used to type HLA-A, -B, -C,-DRB, -DQ and -DP. Six runs of 24 samples and one run of 12
samples were performed comprising a total of 147 samples. The
kit manufacturer’s protocol was followed and all library
preparation was performed manually. Samples comprised thefollowing: 73 local DNA; 54 UCLA reference DNA; and 20
blinded samples kindly provided by the Transplant Immunology
Laboratory at Baylor Scott and White Hospital, Dallas, USA. The
local and UCLA DNA samples were selected to cover a range ofHLA genotypes commonly seen in the New Zealand laboratory,
as well as some rarer alleles.

With the manual library preparation method average library yield
was 1.18 ng/µL (0.22-2.42); average cluster density was 946
K/mm2 (242-1529); average clusters passing filter was 94.3%
and average reads > Q30 was 95.4%. Average read depth using
the manual method is shown in Table 2 and was greater than
200x for all loci.

OBJECTIVES
Very high resolution typing of HLA genes by Next Generation
Sequencing (NGS) methods is becoming mainstream in
histocompatibility laboratories for the workup of haematopoietic
stem cell and solid organ transplant patients and donors.
Determining the full sequence for class I and extended exon
coverage for class II genes produces HLA types with far fewer
ambiguities than traditional Sanger sequencing based typing(SBT) and other lower resolution typing methods. In addition
NGS brings benefits of higher throughput and lower cost.

NGS was performed using the Illumina MiSeq platform andresults were analysed using TruSight Assign, (supplied with theIllumina TruSight kit). NGSengine (GenDx) was also used fordata analysis, as a comparison.
Concordance with the known genotype to the three field level
was assessed. In addition, library yield, cluster density, %clusters passing filter, % reads>Q30 and average read depth
were examined.

This poster describes the validation of the Illumina TruSight
HLAv2 HLA typing kit using the Illumina MiSeq instrument andthe Eppendorf epMotion® 5075 liquid handling robot.

Subsequent to the main validation the library preparation
procedures were transferred to the epMotion® 5075 with the aidof Eppendorf and Illumina technical support. The epMotion®
5075 was programmed to process 12 samples at a time for thePCR clean up, normalization, tagmentation and clean up steps.
Just prior to the library amplification step two batches of 12
samples were pooled, so the final library contained 24 samples
for NGS. Table 1 shows the library preparation timeline using theepMotion®5075. Four runs of 24 samples were performed using
the epMotion®5075, using samples previously typed with themanual library preparation procedure.

Table 1: Library preparation on the epMotion®5075.
Method StepTime (mins)

PCR clean up

90 (x2)

Normalization, tagmentation, clean up

140 (x2)

Library amplification

20

Clean up50

ACKNOWLEDGMENTS
Table 2: Average read depth and % genotype ambiguity by locus using the manual method. Thank you to Dr. Medhat Askar andhis laboratory at Baylor Scott andWhite Hospital, Dallas, forLocus
AB
C
DRB1 DRB3 DRB4 DRB5 DQA1 DQB1 DPA1 DPB1
supplying the DNA samples for theAverage
blinded study.
251
260
245
239
238
215
232
237
218
203
214
read depth%
Thank you to all the NZBS Tissue
Genotype 0.00 0.00 0.00 3.40 0.00 0.00 0.00 0.00 0.00 0.00 21.09
Typing staff who supported theambiguity
validation and implementation ofNGS.
Table 3: Concordance between NGS and known genotype using the manual method.
% Concordance

AB

CDRB1

DRB345

DQA1

DQB1 DPA1 DPB1

Local samples

100

100

98.6

98.6

98.6

100

98.6

100

100

UCLA samples

100

98.1

98.1

92.6

100

96.3

100

100

92.6

Blinded samples

100

100

100

100

100

100

100

100

100

Printed by

DISCLAIMER
The authors have no conflicts ofinterest to declare.

As shown in Table 2 no genotype ambiguities were observed
except for the HLA-DRB1 and –DPB1 loci. For DRB1 this wasdue to DRB1*12:01:01 and DRB1*12:10:01 which differ in exon
1, which is not covered with the Illumina kit. For DPB1 there
were a number of ambiguities which could not be resolved, due
to polymorphism outside the NGS amplicon or lack of phase
continuity.
Concordance between the observed and expected genotype tothe three field level as shown in Table 3 was excellent. The lackof concordance for local samples was due to: C* NGS = C*04:82
but ref type = C*04:01 (exon 5 difference); DRB1 NGS givingDRB1*07, *12 but ref type = DRB1*12, 13 (this repeated as reftype); DRB345 NGS giving DRB4/DRB3 whereas the reference
type was DRB3 only (this repeated as the reference type); DQB1
allele dropout of DQB1*04. Lack of concordance with the UCLA
samples was mostly due to the reference typing being pre-NGS.
There was 100% concordance of NGS with the genotypes of theblinded samples.
For the local samples, genotype failure rate (ie. no resultsobtained) varied between zero (HLA-A, -B, -C, -DQB1, -DPA1
and – DPB1) and 2.8% (DQA1). Failure rate in the UCLAsamples was higher (0-7.4%) possibly due to poorer quality
DNA. There were no failures with the blinded samples.
During the validation the library preparations performed on theepMotion®5075 gave much higher yields than the manual
method (average: 13.8ng/µL), however the cluster density waslower (average: 541.3 K/mm2), average read depth was lower
and failure rate was higher. These findings led to the use of anincreased amount (2.5x) of library product for the sequencing
procedure, with this change the NGS results with theepMotion®5075 have been as good as the manual method. Useof the epMotion®5075 allows up to four runs of 22 samples perweek to be typed by NGS.

CONCLUSIONSThe Illumina TruSight HLAv2 next generation sequencing kit was
shown to be highly effective at performing high resolution HLA
typing for transplant patients and donors as well as other clinicalneeds. Typing is achieved at a three field resolution or better inmost cases without the need for further time consuming andcostly testing. The use of the liquid handling robot reduces
variability between technicians, minimizes the risk of errors andalleviates the issue of extreme manual handling stress.