Amplicon-EZ Frequently Asked Questions

Amplicon-EZ is a fast, cost-effective sequencing service for purified PCR products between 150-500 bp. It uses Illumina® short-read sequencing and is ideal for targeted applications like SNP genotyping and clone verification.

Submit purified PCR products (150–500 bp) with or without partial Illumina® adapter sequences. Each sample should contain at least 500 ng of double-stranded DNA, normalized to 20 ng/µL, and placed in microcentrifuge tubes. For best results, ensure your sample shows a single band on a gel.

This service is ideal for:

• SNP genotyping
• Genome editing clone verification
• Somatic analysis
• Complex variant discovery

• Use purified PCR products sized 150–500 bp
• Ensure ≥500 ng of double-stranded DNA per sample
• Normalize concentration to 20 ng/µL
• Use water, EB, or low TE buffer (<0.1 mM EDTA)
• Quantify using Qubit™ or PicoGreen™ (avoid NanoDrop)
• Submit samples in labeled microcentrifuge tubes or 96-well plates matching your order details
• Upload a gel image and include your order receipt with the shipment

Yes, GENEWIZ does not adjust concentrations before sequencing. Equal volumes are used for each sample, so normalization is essential.

Samples that do not meet the guidelines may be delayed, which could extend turnaround time.

No, Amplicon-EZ only accepts products within this size range. For other sizes, consider our alternative sequencing services.

Results are typically delivered within 3–8 business days.

Pricing depends on project details. Please submit an order to view applicable pricing.

The Illumina® MiSeq platform, or equivalent, is used for Amplicon-EZ.

For the forward sequencing read: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’

For the reverse sequencing read: 5’-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’

Amplicons with the partial adapters should be constructed as follows:

We suggest designing PCR primers with the adapter sequences added to the 5' end of your primers.

Samples can be purified via DNA-binding beads/columns, enzymatic cleanup or gel purification.

Amplicon-EZ typically delivers around 50,000 sequencing reads per sample.

• If your sample yields 10,000-40,000 reads, we can provide additional sequencing upon request to reach the target.
• If your sample produces fewer than 10,000 reads, it may indicate a sample-related issue based on past troubleshooting experience.

Amplicon-EZ is optimized for samples that produce a single, distinct band within the 150–500 bp range. While samples with multiple bands can be submitted, we cannot guarantee successful library preparation or consistent sequencing results for all fragments. Variations in fragment size can affect sequencing efficiency, which may lead to uneven coverage or missing data for some PCR products.

Yes, all raw data (i.e. FASTQ files) will be delivered upon project completion.

The type of analysis depends on amplicon size and the information provided in "Step 3: Data Analysis" on the order form. See the flow chart below to determine which analysis is available for your samples:

This analysis generates an interactive HTML-based report that includes variant detection (SNPs and INDELs up to 20 bp) as well as genotyping information (homozygous wild type, heterozygous, and homozygous mutant). Unique sequences will also be quantified. Each sample will be represented individually in a user-friendly interface.

This analysis generates results that include a list of unique sequences up to 450 bp and their relative abundance in the sequencing data.

Yes, custom bioinformatics services are available upon request and handled on a case-by-case basis. Examples include:

• De-multiplexing using in-line barcodes
• Sample-to-sample comparisons
• Processing large numbers of amplicons
• Multiple primer pairs and/or reference sequences per sample
• Heavy/light chain analysis

The GENEWIZ NGS team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation. You can contact the team by submitting an inquiry.

Our team prioritizes fast turnaround times and provides a timeline based on first-pass processing. The turnaround time listed within the quote is inclusive of all steps quoted, unless otherwise noted. If repeat processing is required, the turnaround may be subject to change and will be proactively communicated to the client.

If the scope of a project changes after project initiation or if sample or project clarification is required after sample receipt, GENEWIZ may reassess the turnaround time based on the subsequent communications and modifications (if applicable).

All extraction kits and reagents are routinely updated to remain best-in-class. If you are seeking access to previous versions of our method, please submit an inquiry (availability is assessed on a case-by-case basis).

Extractions are performed to the best of our ability, and we are unable to guarantee yield due to multiple variables that may affect sample yield and quality. Costs to cover the work performed will be applied, regardless of the outcome.

All preparation kits and reagents are routinely updated to remain best-in-class. If you are seeking access to previous versions of our method, please submit an inquiry (availability is assessed on a case-by-case basis).

For most of the services, an initial sample QC is included within the fee of the project for all samples that will proceed to the next processing stage (e.g. library preparation or sequencing) unless otherwise noted within the quotation. Resubmissions or additional, optional samples may incur a nominal fee for QC.

Initial sample QC can include:

• Assessing RNA concentration and integrity
• Assessing DNA concentration and DNA size (for select projects)
• Assessing premade library size and concentration
• Assessing cryopreserved cells by cell count and viability

The sequencing platform will be listed within the Service Description of the quotation.

Unless specifically noted in the quotation, GENEWIZ reserves the right to choose between equivalent instruments depending on the target read depth and configuration requested. If a specific instrument is required, please add special comments in your quote/order and notify the GENEWIZ NGS team prior to project initiation.

You can contact the GENEWIZ NGS team at:

• US: NGS@azenta.com
• EU & UK: NGS.Europe@azenta.com

Illumina-based projects

For samples that pass QC and libraries prepared at GENEWIZ:

Data Quality

• NovaSeq 2x150bp: ≥85% of bases ≥Q30
• NovaSeq 2x250bp: ≥80% of bases ≥Q30
• MiSeq 2x150bp: ≥80% of bases ≥Q30
• MiSeq 2x250bp: ≥75% of bases ≥Q30

Data Yield

• Within 10% of total data target yield per lane or flowcell, unless otherwise noted
• Within 20% of per sample target yield, unless otherwise noted

Quality and yield for samples that do not pass QC and are processed at best effort are not guaranteed. Premade libraries/library pools submitted for sequencing only quality and yield are evaluated on a case-by-case basis.

Multiplexing is performed to the best of our ability to ensure relatively even data distribution amongst samples.

PacBio-based projects

Due to various sample-related factors that may influence long-read sequencing yield and quality, GENEWIZ cannot guarantee overall data output and quality. However, based on extensive experience with long-read workflows, GENEWIZ has established target metrics based on the PacBio system based on the sample submitted and library type. If a project does not meet these targets, a thorough review of the processing will be performed. If the issue is determined to be unrelated to the sample, a repeat or top-off will be performed as necessary.

Due to the possible wide range of performance being influenced by sequence complexity and sample quality, GENEWIZ does not guarantee average coverage or on-target specificity for each sample. We can instead recommend target data output that would increase the chances of obtaining the desired coverage based on the sample and library type utilized.

Data analysis is performed to the best of our ability and results are not guaranteed for samples or libraries not passing QC or do not fit the analysis pipeline’s criteria.

Yes, we can recommend the best data delivery option based on the platform and project details. Options include:

• Secure File Transfer Protocol (sFTP) - additional charges may apply
• Customer Cloud Account - AWS, Microsoft Azure, Google Cloud
• Hard Drive - additional charges may apply

Delivery to most customer cloud solutions is free of charge. If you have any questions, please submit an inquiry.

By default, results are sent via a secure File Transfer Protocol (sFTP). Please refer to our sFTP Data Download Guide for instructions on how to download your data and troubleshooting tips. Additional charges may be applicable for large data transfers.

Please refer to the delivery email sent from GENEWIZ for detailed information on the transfer and consult with your IT department to ensure compliance with your institution’s policies.

We take data security very seriously and we make every attempt to keep your data private and protected. The data transfer we offer is secure; however, should you desire an alternative delivery method, we are happy to work with you.

We hold any remaining samples for up to 3 months after project completion. Clinical samples processed in our Regulatory-Environment or CLIA-licensed laboratory may have longer sample storage timelines. Please contact us if you would like samples retained for a longer period or shipped back to you.

GENEWIZ offers raw data storage (i.e. FASTQ for Illumina, BAM for PacBio, POD5 for Oxford Nanopore) for up to 6 months after project completion. Data generated for samples processed in the Regulatory-Environment or CLIA-licensed laboratory may have longer data retention timelines. Please contact us if you would like the data to be retained for a longer period.

View our Sample Submission Guidelines for instructions on preparing and sending samples. Organize your samples in tubes or plates following the order indicated on the sample submission form.

• Tubes: Prepare samples in clearly labeled and well-organized 1.5 mL flip-cap microcentrifuge tubes. Please avoid using Parafilm to seal the tubes.

Unless otherwise instructed, GENEWIZ reserves the right to combine multiple vials of the same sample for extraction and/or library preparation.

• Plates: For projects with 16 or more samples, prepare samples in clearly labeled, securely sealed 96-well full-skirted PCR v-bottom plates. Arrange the samples vertically by column (i.e. A1, B1, C1, etc.)

Ship samples directly to our facility. Use the shipping address listed on the order receipt.

At GENEWIZ, we know that samples are valuable and not always perfect. We're here to help find solutions for working with less-than-ideal samples. Please reach out to us by submitting an inquiry, and we’ll work with you to meet your project needs.

For a quick tutorial, watch the video below.

For a quick tutorial, watch the video below.

The status of your order, including an estimated date of delivery, can be viewed anytime through your GENEWIZ account. Visit the Order Summary page of your project to find the current order status.

Option #1: Ship samples directly to our facility. Use the shipping address listed on your order receipt.

Option #2: For double-stranded DNA samples, submit samples into a local GENEWIZ dropbox which are conveniently located throughout the US, Europe and United Kingdom. You can easily locate a dropbox near you by logging into your account and choosing “Select from my dropboxes" during the order checkout process. Place your order receipt and samples in a Ziploc bag packaged according to sample submission guidelines, and place in dropbox for pickup. If you do not have a dropbox near you, please request a new dropbox location here.

• Note: Not recommended for RNA and primary sample types.

During checkout, consult the Order Summary  page to find out the daily cutoff times for your selected dropbox (see example below).

If you miss the dropbox deadline, feel free to ship samples directly to us.