Redefining simplicity in targeted sequencing

Amp-Seq is a mid-plex targeted genotyping by sequencing workflow for agricultural biotechnology genotyping on an industrial scale. Focusing on workflow simplicity paired with automatability, fastest processing time and unparalleled cost effectiveness, Amp-Seq delivers a solution that enables in-house genotyping on an unprecedented scale.

Key benefits

  • Performance - High design success rates and SNP call rates*
  • Simple, easy to adopt workflow. See figure 1 on the workflow tab.
    • Simple and user friendly protocol
    • Minimises impact of user errors
    • Low number of reaction components
  • Cost effective
    • Reducing sequencing costs up to 50%*
    • Reducing labour costs by minimising hands-on time*
    • Low reagent costs and ultra-low reactions volumes*
  • Increased throughput and reduced turnaround time by up to 30%*
    • Industry leading protocol turnaround time - up to 1.8X faster*
    • Same-day-sequencing
    • Pool of thousands of samples and target up to 5,000 markers per sample
    • Compatibility with undiluted crude input material*
  • Reduced environmental impact with ambient temperature stable components
    • Reduce dry ice usage, CO2 emissions and shipping costs  
    • Reduced energy consumption and freezer space requirements 
  • Confidence in supply
    - LGC Biosearch Technologies manufactured reagents
    - Extensive on- and off-site local support resources
    - High-quality standards and manufacturing accreditations

* Validated against the targeted sequencing technologies most prevalently used in the agricultural biotechnology industry. Review the Amp-Seq application note for full details. 

Amp-Seq overview

The proprietary LGC Biosearch Technologies Amp-Seq method was developed in collaboration with the agricultural biotechnology industry to ensure it meets the highest standards in industrial genotyping. The unprecedented workflow simplicity and scalability enables a frameshift in throughput that can drive the pace of breeding programs to exceed expectations in reducing time to bring new traits to the market.

The high SNP call rates and concordance versus alternative methods facilitates transitioning to Amp-Seq while retaining expected data comparability and reproducibility. Additionally, the robustness of the method drives increased evenness of coverage and compatibility with a variety of sample types and preparation methods, which in turn guarantees significant workflow cost reductions.

  • High-throughput workflow for NGS-based genotyping in your lab (in-house)
  • Co-developed with industry leaders for agricultural biotechnology
  • Proprietary chemistry
  • Target up to 5,000 markers per sample
  • Compatible with a variety of sample types and DNA preparation methods
  • The Amp-Seq workflow includes:
    • Amp-Seq targeting oligo library (panel)
    • Amp-Seq core reagent system
    • Amp-Seq index plates
    • BiosearchCaller analysis pipeline
  • The workflow deployment service includes:
    • Targeting oligo library design service
    • Optimisation for various species and sample types
    • Wet-lab implementation support reagent system deployment
    • Automation compatibility and scale up support (optional)
    • BiosearchCaller data analysis pipeline installation and support (optional)
    • Customisation options (optional)

Amp-Seq is developed for high-throughput genotyping applications including:

  • Genomic selection
  • Parentage testing
  • Marker assisted selection (MAS) and back-crossing (MABC)
  • QA/QC testing
  • Plant and animal breeding
  • Other high throughput genotyping applications


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Figure 1. Fast, simple and user friendly 2-stage protocol in 384-well format. The workflow uses pre-plated reagents and room temperature stable pre-plated indexing plates to drive throughput. The workflow is developed to minimise the impact of user errors, for example through the use of identical PCR cycling conditions. With only one enzyme mix, two reagents and a pre-plated index plate, the reagent system is simple and highly automatable to limit hands-on time.

Technical specifications

For a more detailed review of the quality, robustness and performance of the Amp-Seq method please download the application note or start your custom evaluation.

Amp-Seq is a proprietary, multiplex-PCR based amplicon sequencing technology. The below specifications are applicable to the default Amp-Seq workflow. Customisation of the workflow is optional.

  Feature Specifications
Protocol Default protocol Fast 2-stage protocol + pooling and clean-up
BioIT Targeting oligo library design Available as Biosearch Technologies service, default turnaround time 4-6 weeks
Optimised oligo library density Amp-Seq has been validated with up to 5,000 markers
Data analysis pipeline BiosearchCaller data analysis pipeline available for on-premises installation and setup
Variants in flanking sequence Compatible with de-novo variant discovery
Typical sequencing depth Dependent on species and targeting oligo panel
Reagents Core reagent system 3 component system, pre-mixing enables just 2 dispensing steps
Reagent system 'kit' sizes 384 and 3,840 (and multiples of those)
Reagent system stability Minimum 1 year shelf life at -20 °C
Indexing system Conveniently plated combinatorial dual indexing (CDI) system for up to 9,216 samples per lane
Bead re-use reagents Compatible and included by default
DNA Input DNA quantity Typically, 5-10 ng (dependent on genome size and primer pool QC)
HotShot (NaOH) compatibility* Compatible but not included by default
Equipment Thermal cycler compatibility Compatibility data available for Hydrocycler™ and Soellex™, compatibility testing on other platforms through co-development
Sequencer compatibility Illumina (other platforms through co-development and customisation)
Liquid handler compatibility Biomek i7 compatible, compatibility testing on other platforms optional

* Tested for compatibility on a limited number of species and SNP panels, collaborative pilot testing (see ordering information) will deliver genome and panel specific performance

Key features for the Amp-Seq targeted genotyping by sequencing workflow. Note that the workflow is customisable to your needs.

Quality, robustness and performance

For a more detailed review of the quality, robustness and performance of the Amp-Seq method please download the application note or start your custom evaluation.

Quality control

LGC has over 180 years of history as a recognised high-quality company and assay-to-assay consistency is established with the same rigorous quality controls. The assay reagents are optimised to achieve a high percentage of calls from the SNP panel design and a high SNP depth uniformity* for sample-to-sample data. All reagents are subjected to functional assays to establish high standards. Dry indexing primer plates are assayed for both function and for cross contamination

Reagents are optimised during co-development of the targeting oligo library to be robust and suitable for generating high quality, uniform data across a range of sample input types. Newly designed primer pair pools are quality checked for optimal DNA input and optimal primer concentration to complete the robustness of the Amp-Seq assay.

* SNP depth uniformity for Amp-Seq is calculated as the percentage of SNPs with a depth above 20% of the mean depth for that sample.

Assay stability

High-throughput automation of a large set of sample DNA requires that, at some stages in the protocol, 384-well plates of reactions must sit at room temperature for several hours.

Data in figure 2 demonstrates that the reagents for the Amp-Seq assay are very stable at room temperature.

Figure 2. Amp-Seq assay stability at room temperature. Sequencing data quality characteristics for soy samples processed through the workflow at different rates. Set A; reaction mix was incubated for 20 hours at room temperature after stage 1. Set B: reaction mix was incubated for 20 hours at room temperature after stage 1 and after stage 2. Set C; control, all reactions were processed immediately with no room temperature hold for during either stage 1 or stage 2.

The stability of the Amp-Seq reagents allows for flexibility in running this assay at high-throughput due to the convenient handling of reagents. This simplification of the workflow contributes to the reduced turnaround times offered by Amp-Seq.

Targeting oligo library design service

Library design is performed in collaboration between the Biosearch Technologies team and the customer. To initiate a library design project please contact your sales representative.

Software pipelines are used to design specific primer pools and for data analysis. The design software is rigorously tested to ensure that minimal primer interactions will occur, and that background signals are reduced to a level that does not significantly impact on-target mapping of amplicon reads. This level of stringency ensures that sequencing runs are efficient and very little sequence is wasted. Very high first pass design success rates are typically achieved, reducing the total turnaround time required for targeting oligo library design.

BiosearchCaller data analysis pipeline

The BiosearchCaller data analysis pipeline is available for installation and setup in your laboratory. It is designed to detect variants ensuring high variant calling accuracy and high genotype call concordance of diverse sample data when compared to other genotyping technologies.

Protocol automation

Data in figure 3 displays the suitability of Amp-Seq for automation. Consistently high performance was shown across 384 replicates of soy DNA, with a SNP call rate of over 98% and a SNP depth uniformity of amplicon coverage of over 90% on average. The flexibility to automate the workflow without impacting data quality further contributes to the reductions in turnaround times offered by Amp-Seq.

Figure 3. Automation of the Amp-Seq assay. Performance of the soy 1000+ panel illustrates consistently high performance of the Amp-Seq assay when the protocol was automated using a Biomek liquid handler and dried index plates were used.

Robustness – wide range of panel sizes

The robustness of the Amp-Seq assay was assessed with genomic DNA from several crops, including canola, maize, sorghum, soy, strawberry and wheat, using SNP panels of different sizes. Figure 4 illustrates data from maize panels of 1,152, 1,920 and 5,000 SNPs that clearly demonstrates the flexibility and robustness of the assay.

Figure 4. Amp-Seq assay robustness across a range of panel sizes. Maize B73 DNA was assayed with panels of 1,152, 1,920 and 5,000 primer pairs. Sequence data was downsampled to an average of 130.3, 131.1 and 115.7 reads respectively. All panels passed our specification of 90% for percentage of SNPs called and 80% for SNP depth uniformity of amplicon reads.

Robustness – compatible with a variety of sample types- and DNA preparation methods

The robustness of the assay was also demonstrated using crude extracts from maize leaf tissue as input as shown in figure 5. This compatibility with undiluted crude input material allows for increased throughput and turnaround times by reducing upfront labour and logistics costs.

Figure 5. Amp-Seq results for crudely extracted, undiluted sample material. Crude, undiluted maize leaf punch extract was used in the Amp-Seq protocol with a Maize 1,152 SNP panel. The figure shows sequence analysis specifications for: i) percentage of reads that mapped to the correct sequence region (On target SNP %); ii) Percent of the 1,152 SNPs that were detected (SNP %) and iii) SNP depth uniformity (%) of mapped reads for the 1,152 SNPs at an average depth of 134 reads. Quality of data will depend on the exact method of extraction.

Frequently asked questions

We have successfully tested up to 5,000 markers using the Amp-Seq technology.

During development and optimisation of the Amp-Seq workflow, both purified (clean) and crudely-extracted DNA were tested. As detailed in our application note, good results were obtained using a HotSHOT-like extraction involving NaOH and heat. Although Amp-Seq has shown compatibility with undiluted crude input material, the quality of data obtained will depend on the exact method of extraction and optimisation may be required.

The Amp-Seq process is optimised to work with an input of 2.5-30 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.

The Amp-Seq library preparation protocol consists of two amplification stages, with a simple transfer of material between Stage 1 and Stage 2, and may be completed in less than 2 hours. Post-processing in preparation for sequencing, including library pooling and clean-up, size estimation, quantitation, and dilution may take an additional 1.5 to 2 hours.

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

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

Yes, 1X75 sequencing is compatible but please inform us during the primer design process if this is a requirement for you, as primer design can be affected by these short read sequencing protocols. In addition, paired-end sequencing is also compatible.

Species- and panel-specific read depth recommendations are provided during pilot testing. A typical recommended read depth is 150x.

It is possible for libraries to be made for sequencing on alternative platforms to the Illumina systems, but these may need to undergo co-development and customisation. Please enquire for the options and the latest compatibility data.

Yes, we recommend automation of library preparation using a liquid handler to further contribute to the reductions in turnaround times offered by Amp-Seq. Biosearch Technologies have successfully automated this process using a Biomek i7 as shown in our application note, where consistent performance was shown over 384 DNA replicates, a SNP call rate of over 98% and a SNP depth uniformity of amplicon coverage of over 90% on average. As automation with the Biomek i7 had no impact on data quality, we expect automation to be possible with other liquid handling platforms. If you require support with liquid handler compatibility testing, please contact XXX.

We have developed 24 plates of 384 unique combinatorial indexing primer sets for a total of 9,216 possible combinations.

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

Yes, Amp-Seq can be used to discover de novo SNPs, but the discovery is limited to the targeted regions amplified by the designed primers.

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

Biosearch Technologies have internal data demonstrating concordance of Amp-Seq results 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%. If you would like more information about this, please contact

Biosearch Technologies provides an analysis pipeline (the BiosearchCaller software), compatible with the Amp-Seq 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 technology, a range of plant species were tested. These included both diploid genomes (bovine, maize, soy and sorghum) and polyploid genomes (canola, wheat, and strawberry).

Although it is possible to perform the Amp-Seq workflow manually in a standard molecular biology laboratory without specialised equipment beyond a thermal cycler, it was designed as a high-throughput technology optimised for automation using liquid handlers. If you wish to discuss the requirements to run Amp-Seq in more detail, please refer to the manual for a detailed list of equipment requirements and do not hesitate to contact us with any further questions.

At the current time, Biosearch Technologies offers Amp-Seq in the kit format. The workflow includes a custom targeting oligo panel (for which there is a design service), the core reagent system, index plates and an analysis pipeline. Following design of the targeting oligo library, you will receive the kit reagents and dried indexing plates and be able to download the BiosearchCaller software for the analysis stage.

The standard panel design time is 2-3 weeks. 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 panel, Biosearch Technologies 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, Biosearch Technologies have 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.

Species Panel Size Marker Set
Maize 960 Pre-defined
Maize 1,920 Pre-defined
Maize 1,152 Pre-defined
Soy 1,152 Pre-defined
Bovine 199 Pre-defined
  1. Performance – Amp-Seq demonstrates high design success rates and genotype call rates.
  2. Simple, easy to adopt workflow
    1. Simple and user-friendly protocol
    2. Minimises impact of user errors
    3. Low number of reaction components
  3. Cost effective
    1. Reduces sequencing costs up to 50%
    2. Reduces labour costs by minimising hands-on time
    3. Low reagent costs and ultra-low reactions volumes
  4. Increases throughput and reduces turnaround time >30%
    1. Industry-leading protocol turnaround
    2. Same-day-sequencing
    3. Pool of thousands of samples and target >5,000 markers per sample
    4. Compatibility with undiluted crude* input material
      * Please note that use of crude extract with Amp-Seq may require significant optimisation due to variability in quality and yield.
    5. Automatable with liquid handling
  5. Reduces environmental impact by reducing the amount of plasticware needed.
  6. Confidence in supply – Biosearch Technologies-manufactured reagents

Dependent on your current workflow, you can expect a number of cost reductions.

  1. Compatibility with undiluted crude* input material drives direct reduction of labour costs - confirmed by industry partners to save hours versus alternative methods.
    * Please note that use of crude extract with Amp-Seq may require significant optimisation due to variability in quality and yield.
  2. Superior robustness and up to 2X more uniform coverage drives further reduction of required sequencing costs - by 43% per sample. 
  3. In-house manufactured and cost-effective reagent system in ultra-low volumes
  4. Fast protocol turnaround time - In practice this enables same day loading and overnight running of sequencer, in turn reducing the workflow turnaround time by a full day.
  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 the Amp-Seq in your lab. Please do not hesitate to contact us to discuss this further.

If you require support to adopt the Amp-Seq workflow in your laboratory, please make us aware and we can discuss options for this. We have a team of experienced laboratory staff who will be able to help you get up and running with Amp-Seq technology.

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).

Start your Amp-Seq evaluation

To start your Amp-Seq evaluation, please contact your sales representative or request additional information through the form on this page.

Your Amp-Seq evaluation starts with an initiation meeting to introduce you to your team of Biosearch Technologies’ Amp-Seq experts who will guide you through the next phases of the technology evaluation process.

  Kick off meeting Pilot 1 Pilot 2 Implementation phase Post implementation support
  • Introduction to your Biosearch Technologies Amp-Seq evaluation team
  • Provide us with your performance requirements
  • Set up pilot projects
  • Design your oligo panel (or select pre-defined panel)
  • Provide your samples to Biosearch Technologies
  • Biosearch Technologies processes your samples
  • Present performance data
  • Test the Amp-Seq Reagent System at your site
  • Present and evaluate the performance data
  • Set-up workflow at your site
  • Set-up supply agreement
  • Support wet-lab implementation, installation of analysis pipeline, automation setup (optional)
  • Technical support for protocol, analysis pipeline and automation
  • Customisation of wet-lab chemistry/analysis pipeline (optional)
Timeline 1-3 days 8-10 weeks 4 weeks Variable Variable

* Total timeline is typically 12-14 weeks


Ordering information

The Amp-Seq Reagent System includes:

  1. Amp-Seq targeting oligo library (panel)**
  2. Amp-Seq core reagent system**
  3. Amp-Seq Indexing Plates

** Reagent #1 and #2 combined are also available as a bundled kit. Please enquire below.

The targeting oligo library and the core reagents are available in bundled kits for 384 or 3,840 reactions (or multiples thereof). Indexing plates are ordered separately and are pre-plated in 384-well plates.

Product code Name Reactions Type  
GEN-9900-021 Amp-Seq Reagent System incl. targeting oligo library 384 Bundled kit  
GEN-9900-022 Amp-Seq Reagent System incl. targeting oligo library 3,840 Bundled kit  
GEN-9902-001 through -024 Amp-Seq Indexing Plate 1-24 384 per plate Index plate  

Request a quote or speak to an expert for more information.