personalising diagnostics

AimNext-generation sequencing (NGS) technology has great potential for the futureapplication of HLA typing in routine diagnostic purposes. NGS sequencing allowssequencing of single DNA molecule sequences. This means that the paternal andmaternal alleles can be uniquely identified. Currently, various NGS platforms areavailable on the market with each platform being based on a different technology andeach technology in need of their own requirements to make them compatible forHLA typing. At GenDx we have developed a full NGS workflow from targetgeneration to data analysis for platforms such as Illumina MiSeq, Ion Torrent PGMand PacBio RS Sequencer. Here we would like to show you our latest developmentsfor the Ion Torrent PGM platform.

Materials & methodsWe developed a NGS workflow for the Ion Torrent PGM platform that enables high-resolution HLA typing by NGS. In general the workflow can be described in five steps;

Target generation. The HLA locus-specific DNA sequences were amplifiedusing the NGSgo®-AmpX kit (GenDx) that specifically amplifies the differentHLA genes. After amplification, the locus-specific amplicons were pooled.

Library preparation. The fragmentation and end repair of the amplicons wereperformed in a single reaction, creating blunt-end DNA fragments that weredirectly ligated to the barcode-labelled X adapter and the ISP compatible Padapter. After ligation, bead size selection was performed using AMPure XPBeads to remove small fragments and adapter dimers and to select forfragments of an average size of 400bp. Separate libraries were pooled inequimolar concentrations and the final library concentration was determinedusing KAPA (Biosystems).

Clonal amplification. Using the Ion One Touch 2 system and Ion PGMTemplate OT2 400 kit, DNA fragments were bound to Ion Sphere Particles(ISPs) with the P adapter. Subsequently each DNA fragment was amplifiedusing adapter specific primers and properly amplified ISPs were selected.

Sequencing. The sample was prepared using the Ion PGM Sequencing 400 kitand a 316 v2 chip with a capacity of 3 million reads was loaded manually.After the PGM sequencer was initialized and the chip was applied, thesequencing run was started.

Data analysis. The generated data was imported in NGSengine, a softwarepackage that is developed by GenDx for analysis of NGS data consisting ofHLA sequences. The software is NGS platform independent and canseparate sequences of each locus from the other loci to form anunambiguous typing result.

ResultsWe have taken 18 samples of a standardized genomic DNA reference panel and foreach sample determined the typing result for 5 loci; HLA-A, -B, -C, -DRB1 and -DQB1.Amplification of all loci was successful as seen in Figure 1. Weaker DRB1 ampliconscould successfully be typed. The complete workflow including library preparation andsequencing (Figure 2) was achieved in 3 days and included 6 hours of hands-on time.The NGS data was analyzed using the NGS platform-independent NGSengine®software, developed by GenDx for HLA sequence analysis (Figure 3). An average readlength of 279 bp and an average read depth of 1184 was achieved resulting incoverage throughout the gene for each locus/sample (Fig. 3). A correct typing resultwas achieved in 95% of all cases: 100% for HLA-A, 94% for HLA-B, 94% for HLA-C,94% for HLA-DRB1 and 94% for HLA-DQB1 (Figure 4).

Krol L., Adema J., Kooter R., Ruzius F.P., Bouwmans E.E., Rozemuller E.H., Penning M.T., Van de PaschL.A.L., Westerink N., Mulder W.

GenDx, Yalelaan 48, 3584 CM Utrecht, the Netherlands

Accurate HLA typing by NGS using theIonTorrent PGM with a platform specificdeveloped and tested workflow

Figure 1. A schematic overview of the genomic region ofeach locus that can be amplified using the NGSgo-AmpX kit(A). The agarose gel image shows the successfulamplification of HLA-A, -B, -C, -DRB1 and -DQB1 for 18samples using the NGSgo-AmpX kit (B).

Figure 3. The overview window of NGSengine withanalysed data from two samples shows the correcttyping result without exon mismatches (A). An exampleof a dataset for HLA-A is shown with the genomicoverview and statistical analysis performed byNGSengine (B). Correct typing, complete phasing and fullcoverage throughout the entire gene was achieved withan average read length of 286bp and good sizedistribution. A clear allelic distribution was also obtained,with heterozygous positions having a close to 50/50representation and noise levels being below 10% (B).

Figure 4. The average mappability, read length, read depth andnumber of reads for each locus is represented of the total of 18samples that were taken along with 5 loci. Also shown is theconcordance of the typing results with previously obtainedSanger-SBT results.

Figure 2. An overview of the full workflow for HLA typingwith the Ion Torrent PGM. The total workflow takes 3 daysto complete with about 6 hours of hands on time in total.

Conclusions

The availability of different NGSworkflows allows the end-user tochoose the most preferred HLAtyping NGS platform that fulfills theneeds and requirements of theirlaboratory for their specificapplications. Here we demonstratethe NGS workflow developed byGenDx for the IonTorrent PGMplatform to be a powerful method,generating reliable, accurate andhigh-throughput HLA typing bymeans of NGS.

1

2

3

4

5

Sequences of matching alleles

All reads are shownand can be examined in detail if necessary

One phasing region tocover the entire gene

Allelic distribution withblue and red each

representing one of thealleles

Overview of read depthwith heterozygous

positions indicated withcolor

Read depth(average - max)

Read length(min - max)

Mappability

# possible genotypesand typing result

Exon / Intronmismatches

Phasing regions

©2014 GenDx ASHI-2014-04

ASHI posters 2014-screens_wetenschappelijk posteer inches 15-10-14 10:47 Pagina 4