NGSgo® Full Workflow Illumina

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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).
NGSgo Workflow for Illumina is compatible with Illumina NGS platforms and has been validated on the Illumina MiSeq. NGSgo products are also available for the Ion Torrent and PacBio NGS platforms.

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.

What is NGSgo-LibrX and NGSgo-IndX?

NGSgo®-LibrX contains all the enzymes and buffers to create libraries from HLA amplicons. This includes fragmentation, end-repair, dA-tailing, adapter ligation and barcoding with indices.
NGSgo-IndX contains a panel of NGS platform-specific adapter and indices for barcoding of the libraries, which is needed for sample identification. NGSgo kits are available for multiplexing 24, 96 or 384 libraries in one run.

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 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 catalog 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.

How do I quantify my amplicons?

We recommend using the Qubit fluorometer for amplicon quantification.

In our experience, we obtain similar concentrations for the different loci and for multiple samples, when using the same DNA input for all PCR reactions. We recommend examining all amplicons on a 1% agarose gel to see whether all amplicons show a similar band intensity. If similar intensities are observed we randomly pick ~3 amplicons for each locus that we quantify using Qubit.

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

No, ExoI/SAP purification is not needed.

How do I quantify my libraries?

We recommend using the KAPA library quantification kit for Illumina from KAPA Biosystems to measure the concentra-tion of the library. This is a qPCR-based method, using primers that are specific for the Illumina indices. It contains a set DNA standards of a known concentration to measure the exact amount of functional library in your library. The input for the MiSeq needs to be determined accurately to achieve an optimal cluster density.

It is not necessary to quantitate each individual library when they are made within the same NGSgo workflow.
At the end of the library preparation, all samples are pooled together. This single library pool can then be quantified by qPCR.

How do I clean up my samples?

Magnetic SPRI beads are used for the clean-up and size-selection of the libraries. SPRI beads are not included in the NGS-go kit. We have validated the use of AMPure and Macherey-Nagel SPRI beads.

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

The FastQ files can be analysed with the software package NGSengine. This is a dedicated software package for NGS HLA typing.

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 long can I store the NGSgo-AmpX amplicons?

We recommend to store amplicons or amplicon pools at 4oC – 8oC 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.

How long can I store NGSgo libraries of the Illumina workflow?

We recommend to store the library pool (undenatured) at 4oC – 8oC to prevent freeze/thaw cycles. The libraries are stable for at least 4 months when stored in the refrigerator. Please ensure that the plate or tubes are closed properly to avoid evaporation. Before starting the NGS run, we recommend to (re-)measure the library concentration by qPCR for accuracy. It is also recommended to check the library integrity after long-term storage on an agarose gel. In case the libraries have (partially) degraded, the fragment size will be smaller and the library concentration will be lower.

Do you have Material Safety Data Sheets for NGSgo?

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