Cutting edge technology is essential for offering exceptional genotyping services. In 2013, as the world’s first HLA typing lab, we started putting the breakthrough NGS technology, into routine use for HLA typing in a truly high throughput and highly parallelised fashion. Using this process, over 1 million potential stem cell donors are currently typed per year. In addition to NGS technologies, we still use legacy SSP and Sanger sequencing for specialised applications.
Next generation sequencing
NGS is a catch-all term for a collection of deep, high throughput and parallel sequencing technologies developed to replace the Sanger method, which dominated sequencing until the early 2000s. All NGS technologies allow massively parallel analysis that provides extremely high throughput at much reduced costs.
The NGS platform of choice at DKMS Life Science Lab is Illumina MiSeq and HiSeq sequencing. It is today's most successful NGS sequencing platform, using a 'sequencing-by-synthesis' approach where individual fluorescently labelled nucleotides are sequentially added to template DNA strands. After each cycle of DNA synthesis, the nucleotide specific light emissions are captured, and then converted into raw sequence information by advanced image processing software
Our lab´s current generation of Illumina HiSeq 2500 instruments yields up to 1.2 billion sequence reads per run per instrument. To match the extremely high throughput typical for this NGS platform, we developed neXtype, a piece of custom NGS HLA typing software (Lange et al. 2014). The software is highly scalable and implements an innovative, decision-tree-based allele matching algorithm. neXtype uses sophisticated scoring procedures to automatically assess the quality of typing results, thus minimising the need for user interaction.
Our NGS Workflow and neXtype analysis:
Full length HLA gene characterisation by dual redundant reference sequencing
In addition to the routine high throughput HLA assay based on the short amplicon NGS approach, we developed whole-gene assays to sequence the HLA loci A, B, C, DQB1 and DPB1 from 5' to 3’UTR. Our dual sequencing strategy for full length HLA gene characterisation is based on single molecule real time (SMRT) sequencing on a PacBio RS II and shotgun sequencing on an Illumina MiSeq instrument. Both approaches provide high quality consensus sequences with very low error rates.
However, the risk of sequencing errors increases when characterising full length genes due to the marked increase in sequence length. To achieve zero-error reference-quality sequence characterisation we use both sequencing methods independently on each sample. The completely different technologies complement each other: SMRT sequencing delivers fully phased sequences independent of length, while shotgun sequencing delivers highly accurate sequences even for long homopolymer stretches.
The application of both methods eliminates each method’s systematic errors in the final sequence. With our dual sequencing strategy we have characterised and submitted 600 novel alleles thus far, doubling the number of fully characterised alleles in the IMGT/HLA database. This effort led to the development of two software tools: DR2S for dual sequence analysis and TypeLoader for semi-automatic allele submission to the ENA and IMGT/HLA databases.
Single molecule sequencing
The current generation of NGS technologies is limited by relatively short read lengths. With read lengths of several thousand base pairs, emerging technologies for single molecule sequencing promise to overcome this limitation. While still experimental, these technologies may allow a shift from the current exon based HLA typing approach to a whole gene based typing approach, thus achieving unprecedented typing resolution.
We are actively exploring the potential of two promising technologies, namely single molecule real time (SMRT) sequencing by Pacific Biosciences and Nanopore sequencing (MinION and PromethION) by Oxford Nanopore Technologies. We already use the SMRT technologies routinely for full length characterisation of novel or partially known HLA alleles.
This established capillary based sequencing technology is still used where CE-IVD labelling is required.
Sequence specific primed PCR (PCR-SSP)
HLA typing via sequence specific PCR primers is a flexible and still common method, used mostly with clinical samples where CE-IVD labelling is required.