Amp-Seq One Services FAQs

We have successfully tested up to 5200 markers using the Amp-Seq One technology. Data for a high-density panel (wheat) is available in our application note.

The Amp-Seq One process is optimised to work with an input of 2-30 ng of purified genomic DNA. Optimal input amounts are determined for each new panel, and panel-specific recommendations will be provided. We typically recommend around 5-10 ng of input DNA. The amount needed is dependent on genome size and the primer pool QC

Yes, the Amp-Seq One workflow has been validated in a bovine panel of 199 targets. Specifically, the Amp-Seq One technology successfully validated 22 ISAG bovine samples from more than 10 breeds for the parental heritage information.

The Amp-Seq One library preparation protocol requires only one PCR stage and may be completed in 1 hour. Post-processing in preparation for sequencing, including library pooling and clean-up, size estimation, quatitation and dilution may take an additional 1.5 to 2 hours.

Currently, we are unable to support human clinical diagnostic applications.

Yes. The targeting oligo library design service supports short insertions and deletions (indels) up to 10 bp by default. For longer InDels a custom design is required which may impact design costs. Please enquire with your LGC project manager during the design process what the options are. BiosearchCaller, the Amp-Seq One data analysis software, can call genotypes for single nucleotide polymorphisms (SNPs) and for short (≤10 bp) insertions and deletions (Indels) from both diploid and polyploid species.

Yes. BiosearchCaller, the Amp-Seq One data analysis software, is able to call genotypes for multi-nucleotide polymorphisms (MNPs).

Yes, the panel can be modified to add or remove SNP targets. This would need to be done in collaboration with LGC to ensure that your specific requirements are met.

LGC has a large number of internal datasets demonstrating concordance of Amp-Seq One results with Amp-Seq, which in turn has been tested for concordance with alternative technologies including Flex-Seq. We tested a panel of diverse samples with Flex-Seq and Amp-Seq with a concordance of 98.82%. Concordance between Amp-Seq and Amp-Seq One is 98.19%. If you would like more information, please contact techsupport@lgcgroup.com

LGC provides an analysis pipeline (the BiosearchCaller software), compatible with the Amp-Seq One technology, to produce genotypes from the sequencing data. The software can be accessed via a cloud system and our technical team will support the deployment and initial setup.

During development and optimisation of the Amp-Seq One technology, a range of plant species were tested. These included both diploid genomes (maize, soy, bell pepper) and polyploid genomes (wheat).

The first step is the DataPrep, which qualifies the targets based on designability, and takes about a week. The second step is the actual design of the primers, which takes about a week. The standard design process includes multiple rounds of iterative loops to maximise the design rate. The recovery step, one week, is only applied to handle the hard targets to meet high design rate expectations. Customers are notified at the end of each step. We typically recommend submitting 20-30% more targets than are desired in the final panel to ensure that your final panel meets your requirements.

To design an Amp-Seq One panel, LGC requires a reference genome and the desired targets information, such as chromosome, target position, and expected alleles. This information can be provided in the Data Submission Form. If possible, prioritisation of the targets is helpful as we can work to maximise the design rate for the priority 1 targets. To test designed primers, we also require 380 DNA samples and a bulk DNA sample for oligo QC.

Ideally, information regarding desired panel targets and the DNA samples that you provide for primer testing should be provided using the data collection form. Guidance on how to format and submit information is provided in the form.

Yes, we understand the importance of proprietary genome information and can assure you that any genome information that you submit will remain proprietary. It will be linked with your customer account only and as required can be covered under an existing or new non-disclosure agreement (NDA).

Sometimes it is not possible to achieve a viable design for a desired target. There are several reasons why this can occur. Complexity of the genome and the target region Overlap with the neighbouring targets Primer-dimers potential between oligos Known polymorphism/variability on the genome.

Sometimes, despite a successful primer design for a target, genotyping calls are not achieved for a specific target. This can be caused by several issues including poor primer design and issues with the synthesis of the primers. In addition, if genomic reference data for both parental strains is not provided for target primer design, some primer pairs might not work for all F1 samples.

Missing genotypes are generally attributed to low depth. Certain targets cannot be amplified because of sample quality issues or unknown parental bias in the genomes. Our standard design process addresses the parental bias by avoiding primers on known variability in the genome. Known variation information can be provided to us in the target flanking sequences through the Data Submission form.

Yes, LGC has a small number of pre-defined panels that can be used for evaluation studies. These are listed in the table below. Pilot studies may be performed using either custom- or pre-defined panels.

See the full list here.

LGC uses the benefits of the Amp-Seq One technology to enable cost effective service provision.

1. Amp-Seq One eliminates the requirement for liquid handling equipment to transfer material between reaction stages. This halves your labor and consumable costs as well as reducing capital expenses and maintenance costs (no liquid handler required for stage-1 to stage-2 liquid transfer).
2. Fast protocol turnaround time - In practice this enables same day loading and over-night running of sequencer, in turn reducing the workflow turnaround time by a full day.
3. Superior robustness and up to 2X more uniform coverage drives further reduction of required sequencing costs - by 43% per sample.
4. Room-temperature storage dried down index plates reduce freezer space and (energy) costs as well as prevent the need for an extra plate (consumbles cost). Additionally, the in-house manufactured and cost-effective reagent system in ultra-low volumes.
5. Maximise data output per $ - high design success rates, SNP call rates.
6. Enables pooling of thousands of indexed samples and targeting of >5,000 markers per sample.

Yes, we welcome enquiries to try Amp-Seq One technology in your lab. Please do not hesitate to contact us to discuss this further.

The standard turnaround time for design of a new targeting oligo library is 4–6 weeks (2–3 weeks of in silico design time and 2–3 weeks of synthesis and QC).

Panels designed for Amp‑Seq are compatible with Amp‑Seq One technology as demonstrated in our application note. Both plant and livestock panels developed for Amp‑Seq perform comparably when used with Amp‑Seq One. Some optimization of DNA input amounts and primer concentration may be required for optimal performance.