Efficient Solution for Automated TruSight HLA v2 Sequencing Panel Library Preparation

The major histocompatibility complex (MHC) cell surface proteins on chromosome 6 (Figure 1) are essential for encoding the human leukocyte antigen (HLA) proteins. Besides regulating the human immune system, these genes play a key role in drug response, infectious disease, cancer, autoimmune disease, and transplant rejection.

Characterizing these genes with traditional techniques is a challenging task as they are among the most polymorphic proteins in the genome.

However, these genes can be fully sequenced and characterized with high accuracy using next-generation sequencing (NGS) compared to traditional methods like serological typing, sequence-specific primers (SSP), sequence-specific oligos (SSO), and capillary electrophoresis sequencing (SBT). This leads to the widespread adoption of HLA gene sequencing by NGS.

Gene map of the human leukocyte antigen (HLA) region.

Figure 1. Gene map of the human leukocyte antigen (HLA) region.

The Illumina TruSight HLA v2 Sequencing Panel is a simple and reliable DNA-to-report solution developed to create NGS libraries for use in high-resolution HLA typing. The capability of workflow automation on  Tecan’s Freedom EVO® NGS workstation enables 11 HLA loci (Class I HLA-A, -B, -C; Class II HLA-DRB1/3/4/5; -DQA1, -DQB1, - DPA1, -DPB1) to be sequenced for up to 24 samples, reducing the risk of human errors and cross contamination during library preparation.

Tecan’s TouchTools™ software interface (Figure 2) is user-friendly and assists the operator in library preparation and instrument set-up, thus creating highly reproducible, sequencing-ready libraries through minimized training and reduced operator-to-operator variability.

Table 1. Library preparation throughput on the Freedom EVO NGS workstation

Samples

Amplicons

Reactions

Throughput

6

8

48

3/4 workday

12

8

96

1 workday

24

8

192

2 workdays

Tecan’s TouchTools user interface enables simple selection of sample number and the loci to be amplified.

Figure 2. Tecan’s TouchTools user interface enables simple selection of sample number and the loci to be amplified.

Experimental design

A Tecan Freedom EVO NGS workstation was used to perform the liquid handling steps. Figure 3 shows the deck layout of the Freedom EVO NGS workstation set-up for the TruSight HLA v2 Sequencing Panel. The steps include:

  • HLA PCR amplicons
  • Bead cleanups
  • Normalization and tagmentation
  • Pooling
  • PCR set-up

Deck layout of the Freedom EVO NGS workstation set-up for the TruSight HLA v2 Sequencing Panel.

Figure 3. Deck layout of the Freedom EVO NGS workstation set-up for the TruSight HLA v2 Sequencing Panel.

Figure 4 shows the complete workflow. PCR amplification was carried out in an offline thermocycler for optimizing the workflow. The sophisticated air displacement pipetting technology used by the platform allows precise eight-channel pipetting from 1,000 μl to 0.5 μl.

The three INHECO CPAC thermal devices featured in the platform allow the regents to be maintained cool and optimal conditions to be achieved for the enzymatic steps. They include a Robotic Manipulator Arm, a 96-position magnetic plate separator (Alpaqua® 96S Super Magnet), and an INHECO Thermoshake heated shaker. Moreover, longer unattended runs can be achieved with the compact worktable, which can accommodate up to 12 tip boxes.

DNA size distribution of an example library constructed with the TruSight HLA v2 Sequencing Panel shows a size of ~1700 bp.

Figure 4. DNA size distribution of an example library constructed with the TruSight HLA v2 Sequencing Panel shows a size of ~1700 bp.

Libraries were created for all existing amplicons by running reference samples (n=24) obtained from peripheral blood DNA. A BioRad Experion™ system and a DNA 12K analysis kit were used to perform quality control of the constructed libraries (Figure 4).

An Illumina MiSeq™ Sequencing System used a 300-cycles reagent kit on a 2 x 150 bp run to pool and sequence the samples, with a micro flow cell. The guidelines outlined in Illumina's TruSight HLA v2 Reference Guide1 were followed to perform the qualification protocol for automated TruSight HLA with an input of 400 ng of DNA.

Analysis and results

The sequencing data collected from all loci of the 24 sequenced samples was analyzed using the Conexio-powered TruSight HLA Assign 2.0 analysis software2.

The automated TruSight HLA v2 Sequencing Panel workflow. Timings are indicated for 12 samples with 8 amplicons each, running in less than one working day.

Figure 5. The automated TruSight HLA v2 Sequencing Panel workflow. Timings are indicated for 12 samples with 8 amplicons each, running in less than one working day.

The alleles determined for all samples showed good agreement with the known sequences (Table 2), demonstrating the effectiveness of the HLA typing results provided by the automated construction of libraries on the Freedom EVO NGS workstation.

Table 2. Alleles identified in five of the 24 sequenced libraries prepared using the TruSight HLA v2 Sequencing Panel on the Freedom EVO NGS workstation. In HLA alleles nomenclature, the first number (02:01:01) corresponds to the allele group, the second number (02:01:01) corresponds to the specific HLA allele subtype and the third number (02:01:01) corresponds to synonymous DNA substitutions within the coding region. DRB1 variants are linked to either one or none of the genes DRB3, DRB4 and DRB5, reason why they are not always shown in the sequencing results.

Alleles identified in five of the 24 sequenced libraries prepared using the TruSight HLA v2 Sequencing Panel on the Freedom EVO NGS workstation. In HLA alleles nomenclature, the first number (02:01:01) corresponds to the allele group, the second number (02:01:01) corresponds to the specific HLA allele subtype and the third number (02:01:01) corresponds to synonymous DNA substitutions within the coding region. DRB1 variants are linked to either one or none of the genes DRB3, DRB4 and DRB5, reason why they are not always shown in the sequencing results.

Sample cross-contamination was not observed in homozygous samples (Table 2, rows 4 and 5) during the run. The creation of high-quality libraries was confirmed by sequencing metrics, as shown by the sequencing metrics given below:

  1. Achieved good levels of sequencing coverage with an average of at least 198 for all amplicons (Figure 6)
  2. Very high values of up to 92% were shown by the >Q30 report (Figure 7)

Levels of coverage for each of the amplicons. The horizontal line indicates the 100x coverage threshold.

Figure 6. Levels of coverage for each of the amplicons. The horizontal line indicates the 100x coverage threshold.

Q30 values for each of the amplicons. The horizontal line indicates 75% of >Q30 value." src="/image.axd?picture=2017%2f6%2ftecan_June_8_-_680x_.jpg" style="width: 673px; height: 279px;" title="">

Figure 7. >Q30 values for each of the amplicons. The horizontal line indicates 75% of >Q30 value.

Conclusion

The results discussed in this article have demonstrated the effectiveness of the Tecan Freedom EVO NGS workstation for automated library preparation for the TruSight HLA v2 Sequencing Panel. The integrated, easy-to-use TouchTools interface not only simplifies run set-up, but also reduces the risk of errors.

Acknowledgements

Data contributed by Eric Weimer, Assistant Professor, Department of Pathology and Laboratory Medicine, and Ewa Malc, Postdoctoral Researcher, Department of Genetics and University of North Carolina, Chapel Hill, USA.

Produced from materials originally authored by Dr. Enrique Neumann, application specialist at Tecan Switzerland.

References

  1. TruSight HLA v2 Sequencing Panel Reference Guide. Document #1000000010159 v00. May 2016.
  2. Assign 2.0 TruSight HLA Analysis Software Guide. Document #1000000010450 v00. May 2016.

About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Apr 1, 2019 at 5:13 AM

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