NGSgo® Full Workflow Ion Torrent

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What is NGSgo?

NGSgo is a workflow for high-resolution HLA typing by means of Next-Generation Sequencing (NGS). NGSgo consists of reagents for HLA locus amplification (NGSgo-AmpX) and library preparation (NGSgo-LibrX and NGSgo-IndX).

What is NGSgo-AmpX?

NGSgo®-AmpX consists of dedicated primer sets, enabling the amplification of HLA genes. HLA locus-specific amplification is performed in a thermal cycler, using the amplification primer mix, template genomic DNA and the QIAGEN LongRange PCR kit. Each HLA locus is amplified separately. After amplification, the amplicons can be pooled for downstream processing in the NGSgo® workflow for next-generation sequencing.

Do I need to purify my amplicons using ExoI/SAP?

No, ExoI/SAP purification is not needed.

Which HLA loci can be sequenced with NGSgo?

NGSgo®-AmpX products are available for the following HLA loci: HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, DPB1 and HLA-G.
The amplification strategy of NGSgo-AmpX is schematically shown for each HLA locus in the figure below.

How long can I store the NGSgo-AmpX amplicons?

We recommend to store amplicons or amplicon pools at 4 oC – 8 oC to prevent freeze/thaw cycles. The amplicons are stable for at least 3 months when stored in the refrigerator. The amplicons do not have to be cleaned up: you can simply store the PCR reactions. Please ensure that the plate or tubes are closed properly to avoid evaporation.

Can I freeze/thaw the NGSgo kits multiple times?

Yes, the NGSgo kits can resist multiple freeze/thaw cycles without affecting the functional performance. NGSgo-AmpX primers, after reconstitution in water, can be exposed to 25 freeze/thaw cycles. NGSgo-LibrX and NGSgo-IndX kits can be exposed to 16 freeze/thaw cycles.

How many samples can I sequence with NGSgo?

There are different NGSgo products available for low, medium to high-throughput sequencing:

For HLA amplification,
NGSgo-AmpX kits are available for 24, 96 and 384 reactions.

For HLA library preparation,
NGSgo-LibrX kits are available for 48 or 96 reactions.

For HLA library barcoding,
NGSgo-IndX kits are available for 4×24, 2×96 or 1×384 reactions, for multiplexing a maximum of 24, 96 or 384 NGSgo libraries in one NGS run.

See our online catalogue for an overview of all available NGSgo products.

Is NGSgo CE-IVD marked?

The amplification component NGSgo-AmpX (24 rxn) and NGSgo-AmpX (96 rxn) are CE-IVD marked.

The primers for HLA-A ,B, DRB1 and DRB3/4/5 are marked CE 0344
The primers for HLA-C, DQA1, DPA1, DPB1, DQB1 and G are marked CE

NGSgo®-AmpX is also available as RUO

Which thermocyclers can we use for NGSgo-AmpX and which ramp rate?

NGSgo®-AmpX amplification should work on all general thermocyclers.
We have validated the NGSgo®-AmpX reagents using the ABI9700 thermocycler systems using MAX mode which applies a ramp rate of 5oC/s.
Please make sure to use an ´artificial hot-start´ by placing the amplification reaction in a pre-heated (80oC) thermocycler.

What is NGSgo-LibrX for Ion Torrent?

NGSgo®-LibrX consists of all reagents that are needed to create libraries from HLA amplicons. This includes the fragmentation, end-repair, dA-tailing and adapter ligation reagents.

What is NGSgo-IndX for Ion Torrent?

NGSgo®-IndX consists of all available barcoded adapters. There are 96 barcoded adapters in total, so 96 samples can be individually labeled.

Can loci be pooled per sample?

After amplification and before the start of the library preparation, loci can be pooled together for each sample. This significantly reduces the number of samples that are taken along in the library preparation. It is however not advisable to pool HLA-DRB1 and HLA-DRB345 amplicons together because this may cause problems during analysis due to the high similarities between these genes.

How many samples can be performed in a run?

We supply 24 barcoded adapters in our NGSgo®-IndX kit. These adapters can be used to label a maximum of 24 individual samples. With a 316 chip 24 samples (5 loci) and with a 314 chip 6 samples (5 loci) can be sequenced in a single run.

How do I clean-up my samples?

Only one clean-up step is needed in the entire workflow. This step is performed using SPRI magnetic beads.

How do I quantify my libraries?

We recommend using the KAPA protocol from Biosystems. This method by qPCR includes primers that are specific for the Ion Torrent adapters. This way only properly formed libraries are measured. Specific standards are also taken along making it possible to calculate the exact amount of libraries present in your sample.

Do I need to quantify all my libraries?

All samples can be pooled at the end of the library preparation. The library pool can then be quantified by qPCR (KAPA Biosystems). There is no need to quantify individual libraries when they are made in the same workflow. The input for clonal amplification needs to be calculated exactly, therefore the quantification of the library pool is a crucial step.

How do I analyze the NGS data to get HLA typing results?

The data files generated by the Ion Torrent can be analysed with the software package NGSengine. This is a dedicated software package for NGS HLA typing.

Do you have Material Safety Data Sheets for NGSgo?

Yes, the MSDS for NGSgo can be downloaded from here.