Amp-Seq workflow

During development and optimisation of the Amp-Seq workflow，both purified（clean）and crudely-extracted DNA were tested。As detailed in our应用程序编号，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 2.5-30ng of purified genomic DNA。Optimal input amounts are determined for each new panel，and panel-specific recommendations will be provided。We typically recommend around5-10ng 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 Stage1and Stage2，and may be completed in less than2hours。Post-processing in preparation for sequencing，including library pooling and clean-up，size estimation，quantitation，and dilution may take an additional1.5to2hours。

Yes，the Amp-Seq workflow has been validated in a bovine panel of 199targets。Specifically，the Amp-Seq technology successfully validated22ISAG bovine samples from more than10breeds for the parental heritage information。

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

Yes，1X75sequencing 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 i7as shown in our应用程序编号，where consistent performance was shown over384 DNA replicates，a SNP call rate of over98%and a SNP depth uniformity of amplicon coverage of over90%on average。As automation with the Biomeki7had 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 developed24plates of 384unique combinatorial indexing primer sets for a total of 9216possible combinations。

Yes。The targeting oligo library design service support short insertions and deletions（indels）up to 10bp 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 data analysis software，can call genotypes for single nucleotide polymorphisms（SNPs）and for short（≤10bp）insertions and deletions（Indels）from both diploid and polyploid species。

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

是，Amp-Seq can be used to discoverde novoSNPs，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 contacttechsupport@lgcgroup.com。

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 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 possible to perform the Amp-Seqworkflow 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-Seqin 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 is2-3weeks。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 submitting20-30%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 1targets。To test designed primers，we also require380 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
• 覆盖板，覆盖板
• 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 F1samples。

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。

1. 性能，性能–Amp-Seq demonstrates high design success rates and genotype call rates。
2. 简单，easy to adopt workflow
   1. 简单和用户friendly protocol
   2. 用户最低限度
   3. 低强度混凝土
3. 成本，成本
   1. 回收成本提高50%
   2. 回油箱用minimising hands-on time
   3. 低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. 雨水管和target>5000markers per sample
   4. 可压缩性窗框*input material
      *Please note that use of crude extract with Amp-Seq may require significant optimisation due to variability in quality and yield。
   5. 可自动调节窗
5. 可回收的impactby 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 undilted 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-by43%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. 最大数据输出功率$-high design success rates，SNP call rates
6. 已安装的焊缝和targeting of>5000 markers per sample

Yes，we welcome enquiries to try the Amp-Seqin your lab.Please do not hesitate tocontact usto 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 anew targeting oligo library is4-6 weeks（2-3 weeks ofinsilicodesign time and 2-3 weeks of synthesis and QC）。