IntroductionHLA typing by Next Generation Sequencing (NGS) is increasingly being adopted for high-resolution HLA genotyping. Holotype HLA is a commercially available NGS-based HLA genotyping product for up to 11 HLA loci that has been implemented in more than a dozen clinical labs worldwide. To ensure reproducibility and repeatability across technical staff and to reduce the possibility of human error, pre-PCR and post-PCR steps of the protocol can be automated on liquid handling robots due to the flexibility of Holotype HLA.We present the results of Holotype HLA 24/7 and 24/11 as automated on the DX-A liquid handling robot by Texas BioGene (TBG). The DX-A is a cost-effective robot with a small physical footprint that only needs a laptop to operate (Picture 1), and can provide accurate, consistent results eliminating the risk of human error.

MethodsWe performed a Holotype HLA 24/7 run using the automated protocol designed specifically for the TBG DX-A pipetting system on 24 DNA samples that were previously typed manually with the Holotype HLA kit. Each sample was amplified at 7 loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DPB1 and HLA-DQB1). The samples were sequenced on an Illumina Miseq using the 2x250 bp chemistry and the resulting files were analyzed using Omixon’s HLA Twin 1.1.4.2 software.The genotyping results generated from the automated protocol on TBG DX-A were compared to previously determined genotypes from a manually performed Holotype run.

Results & DiscussionThe Holotype HLA protocol in an HLA clinical laboratory to be as efficient as possible - to reduce technician time and opportunities of human error. Additionally, the flexibility of the assay makes it possible to perform the whole protocol manually within about 5 hours of hands on time (HoT). When using the scripts provided for the TBG DX-A the total HoT is reduced from 5 hours to 1:45 hours. With the pre-PCR and post-PCR automation this new protocol can save 3 hours of valuable time and reduce cost when processing Holotype HLA either for 7 or 11 loci. The detailed steps of the protocols, manual and robot hands-on times for the 24/7 kit can be found in Graph 1.

The total time required (including LR-PCR and other incubation times) is about 14.5 hours. During this pilot project, the scripts for the Holotype HLA 24/11 workflow we developed, but the validation is yet to be completed. The hands-on times for the 11 loci kit measured during initial testing are displayed in Graph 2.

The steps that have been automated include: input DNA dilution, LR-PCR setup (Picture 2), amplicon quantitation and normalization, per-sample pooling of HLA loci, library preparation and final pooling. We used both single channel and multichannel (8 channel) pipetting options in the protocol. To leverage the power and accuracy of TBG DX-A automated liquid handling system, we optimized the robot with the least possible number of pipetting steps. The robot run times of both HLA Holotype automated protocols can be seen in Table 1.

ConclusionAutomation of Holotype HLA 24/7 and 24/11 kits on the TBG DX-A was successfully implemented on the TBG DX-A. The protocols that were developed reduced the hands-on time significantly, while the total time of the workflow remains the same as for manual processing. Most importantly, the TBG DX-A eliminates variation and errors among technicians reducing inter-run variability to offer consistency and accuracy in genotyping results.

The TBG DX-A uses a simplified programming language that makes it easy to implement, and easy to modify for different lab throughputs, while being among the most cost effective liquid handling solutions on the market today. All of the above make the TBG DX-A a suitable liquid handling system for use with the easy and streamlined Holotype HLA for any HLA lab.

AUTOMATING HOLOTYPE HLA ON TBG’S DX-ATM ROBOTEfi Melista1, Tina Yu2, Tunde Vago1, Willy Hsu2, Peter Meintjes3, Tim Hague1

1 Omixon Biocomputing Kft2 TBG Biotechnology Corp.3 Omixon Inc

PICTURE 1 - The TBG DX-A is a small, convenient and simple pipetting system.

PICTURE 2 -The layout of TBG DX-A for LR-PCR preparation using Omixon’s Holotype HLA kit.

TABLE 1 - The TBG DX-A pipetting system run times for the Holotype HLA 24 samples 7 loci and 11 loci protocols.

24/7 DX-A total run time 24/11 DX-A total run time

DNA dilution 00:15 00:15

LR PCR preparation 01:35 02:25

Quantitation, normalization, amplicon pooling and cleaning up

01:30 02:05

Library preparation and pooling 01:10 01:10

Total 04:35 05:55

GRAPH 1 - Hands on times for manual (blue) and automated (orange) Holotype HLA 24/7 protocols.

1.7.6 HoT 24/7 Dx-A automation

1.7.6 HoT 24/7

0:15

0:25

0:15

0:15

0:15

0:15

0:20

57.9%

00:45

00:50

00:55

0:55

0:15

0:15

0:20

Step 1 gDNA preparation, Master Mix preparation

Step 2 HLA Class I and Class II Amplification

Step 3 Amplicon Quantitation and Normalisation

Step 4 Library Preparation

Step 5 Size Selection

Step 6 Library Quantitation

Step 7 Sequencing on Illumina MiSeq

Step 8 Data Analysis

Step 1 Step2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8

1.7.6 HoT 24/7 Dx-A automation

00:15 00:25 00:15 00:15 00:15 00:15 00:20 00:00

1.7.6 HoT 24/7

00:45 00:50 00:55 00:55 00:15 00:15 00:20 00:00

Holotype HLA 24/7 Dx-A HoT

GRAPH 2 - Hands on times for manual (blue) and automated (orange) Holotype HLA 24/11 protocols.

1.7.6 HoT 24/11 Dx-A automation

1.7.6 HoT 24/11

0:15

0:25

0:15

0:15

0:15

0:15

0:20

57.9%

1:05

1:001:20

0:55

0:15

0:15

0:20

Step 1 gDNA preparation, Master Mix preparation

Step 2 HLA Class I and Class II Amplification

Step 3 Amplicon Quantitation and Normalisation

Step 4 Library Preparation

Step 5 Size Selection

Step 6 Library Quantitation

Step 7 Sequencing on Illumina MiSeq

Step 8 Data Analysis

Step 1 Step2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8

1.7.6 HoT 24/11 Dx-A automation

00:15 00:25 00:15 00:15 00:15 00:15 00:20 00:00

1.7.6 HoT 24/11

01:05 01:00 01:20 00:55 00:15 00:15 00:20 00:00

Holotype HLA 24/11 Dx-A HoT