AllType™ FASTplex™ – Combining Multiplex PCR with Workflow Simplicity

Whilst most molecular HLA typing methodologies remain relatively static, next-generation sequencing (NGS) has tended to evolve quickly as product manufacturers respond to user suggestions, reagent development and changes in sequencing technology.  One Lambda released AllType™ in Summer 2017: a NGS workflow for all loci, all platforms, all in a single reaction.  This NGS workflow was a game changer, beginning with the long-range PCR of all 11 classical HLA genes in a single multiplexed reaction.  Whilst AllType™ remains a competitive workflow, One Lambda have now released an alternative NGS solution: AllType™ FASTplex™ NGS.

AllType™ FASTplex™ NGS

The rationale behind AllType™ FASTplex™ NGS was to simplify the NGS workflow, save time, and yet still generate high-quality results.  FASTplex™ sets a new standard of simplicity for NGS and is a simple assay that anyone can learn.  Like the classic AllType™ workflow, FASTplex™ begins with all 11 classical HLA loci (HLA-A,B,C,DRB1,DRB3/4/5,DQA1,DQB1,DPA1,DPB1) co-amplified in a single multiplexed reaction (Table 1).  After an initial thermal cycling time of only 2.5 hours, patient-specific amplicons are pooled into a single tube within an hour of commencing library preparation.  This means that approximately 70% of the FASTplex™ protocol is single tube workflow.  The consequences of this are significant: from DNA to sequencing, sample preparation takes less than seven hours and requires only 90 minutes of hands-on time.  An easy single-tube workflow reduces the chances of pipetting error and means that automation isn’t required.  By eliminating most of the pipetting, purification and quantification steps required by ‘traditional’ methods, FASTplex™ significantly reduced the consumables required.  Compared to the classic AllType™ workflow, FASTplex™ uses less than half the plastics, pipette tips and consumables: good news for both operational costs and the environment.  The AllType™ FASTplex™ NGS product also includes paramagnetic beads, further decreasing the cost per test resulting from consumables and third-party reagents.

Table 1: Gene content and coverage for AllType™ FASTplex™ NGS

The FASTplex™ Workflow

The AllType™ and AllType™ FASTplex™ workflows are compared in Figure 1.  FASTplex™ builds on the classic workflow and removes some elements that might be considered undesirable in a non-automated laboratory.  Following amplification and paramagenetic bead purification, sample-specific amplicons are quantified using a Qubit instrument.  AllType™ requires sample-specific normalisation to proceed with 100 ng DNA whilst FASTplex™ allows the user to proceed with 3-30 ng DNA, reducing the pipetting burden as samples ought to fall within this range.  A sophisticated workbook-based tool guides the user through the FASTplex™ workflow, indicating if adjustments are required and performing the necessary calculations.  Unlike the classic workflow where fragmentation, adapter ligation (‘barcoding’) and size-selection are discrete processes, the FASTplex™ workflow simultaneously fragments and barcodes amplicons using a set of ‘FAST’ reagents pre-plated in a 96-well tray; size-selection is not required.  At this point FASTplex™ samples are pooled and the remainder of the workflow occurs in a single tube.  In the FASTplex™ workflow an additional ‘universal barcode’ is ligated to all fragments prior to a secondary PCR amplification.  Thereafter the library is cleaned using paramagnetic beads, quantified and diluted to the concentration required for sequencing.  Working in a single-tube significantly increases the speed and simplicity of the workflow; although bead-based purification and quantitation is still required the volume is significantly reduced compared to e.g. 24 samples.

Figure 1: The AllType™ and AllType™ FASTplex™ NGS workflows compared.

Multi-Platform Support

Both AllType™ and AllType™ FASTplex™ are supported on multiple Illumina platforms and kits, in addition to Ion Torrent platforms.  This provides verified flexibility for the end user, who may choose to utilise a sequencing platform shared by multiple users or acquire their own ‘entry level’ sequencer (e.g. the iSeq 100, with a purchase price of approximately £20,000).  Many sequencing platforms also offer different ‘kit’ sizes which allow users to implement a solution appropriate to their laboratory, i.e. balancing throughput, ‘batch’ cost per sample (considering the sequencing kit) and turnaround time. 

Table 2: Illumina sequencer throughput for AllType™ and AllType™ FASTplex™.

The sequencing platform and kit used also impacts the sequencing time and thus the time taken from sample to HLA genotyping results.  Using the fastest solution, AllType™ FASTplex™ allows the user to obtain HLA genotyping results a mere 26 hours after starting their testing (Figure 2).

Figure 2: AllType™ FASTplex™ is the fastest sample-to-result NGS workflow for HLA.

Download the AllType™ FASTplex™ product sheet

TypeStream Visual™ 3.1: Fast, Fully-Automated Analysis

AllType™ FASTplex™  is supported by TypeStream Visual™ 3.1: intelligent software for the analysis of AllType™ data from Ion Torrent, Illumina and PacBio sequencing platforms.  TypeStream Visual ™ 3.1 retains the familiar (Fusion-esque) ‘front-end’ environment but builds on LIMS integration: SystemLink offer a HistoTrac module that interfaces directly with the TypeStream Visual™ database, and HLA typing data can now be exported to One Lambda’s HLA Fusion™ software with only three clicks.  This allows the integration of high-resolution genotypes with patient/donor records and facilitates antibody tracking and epitope-level analysis using HLAMatchmaker functionality built into HLA Fusion™. 

The TypeStream Visual™ auto-analysis feature streamlines data migration so that data are analysed even when the user is away from the laboratory.  TypeStream Visual™ 3.1 also features an analysis engine that allows multiple samples to be analysed simultaneously, resulting in faster analysis times.  This ensures that AllType™ and AllType™ FASTplex™ are the fastest sample-to-result NGS workflows for HLA genotyping.  TypeStream Visual™ 3.1 offers more user-definable features including health metrics, system comments and the ability to exclude specific gene regions (e.g. micro-satellites).  The analysis experience has been redesigned with a streamlined interface: fewer ‘levels’ of analysis require fewer ‘clicks’ to assign a result (Figure 3).  Upgrading to and maintaining TypeStream Visual™ 3.1 has also been made easier with a result comparison tool that can also be used to verify initial and confirmatory HLA typing data. 

Figure 3: TypeStream Visual™ provides the user with a streamlined analysis experience.

NGS is complicated – but your workflow doesn’t have to be.  Get in touch to learn how you can adopt the AllType™ FASTplex™ NGS method for HLA typing that’s simple, fast and best-in-class.