Back

Amplicon Sequencing Technical Documentation

Technical Details Sample Preparation Data deliverables and file types Troubleshooting Recombination Amplicon size bias Rerun policy

Technical Details

Plasmidsaurus Genotyping Analysis is an amplicon sequencing service that allows you to characterize the allelic composition and frequency at specific loci. By leveraging Oxford Nanopore long-read sequencing, this service empowers you to confidently phase variants and easily sequence extended amplicons (100 bp -25 kb), providing a comprehensive view of your targets. Genotyping Analysis accurately detects all alleles present at >5% frequency.

Sample Preparation

Official sample preparation instructions are outlined on the Amplicon Sample Prep page.

Data deliverables and file types

Data tables for allelic sequences (.tsv) | The “variants” folder reports base call differences, relative to a reference, and their frequencies, for each allele. The “allele-counts” folder contains total counts supporting each allele, displayed in tables.
Virtual gel (.png) | Shows the distribution of read lengths in your sample - great for quickly identifying samples with unexpected PCR products.
Allele sequence files (.ab1) | The “ab1-files” folder contains chromatograms for each allele. The “alleles” folder contains the sequence of alleles in fasta format. Allele-genbank-files provide annotated genbank files for each allele.
Feature annotation and reference alignments | Plasmidsaurus-provided annotations are automatically provided in the feature map (online only). Automatic reference alignments also identify mismatches and associated protein consequences, between your reference and alleles, so you can immediately characterize your genetic variation.
Sequencing statistics including read length and coverage | Histograms characterizing coverage distribution across read lengths as well as other sequencing metrics.
Raw reads | Raw reads mapping to your sample are provided in fastq format.

Troubleshooting

Technical Note: Mitigating PCR-mediated recombination artifacts

Amplicon size bias

What is amplicon size bias?

Amplicon size bias is a phenomenon where shorter DNA fragments are preferentially amplified during PCR, resulting in an underrepresentation of longer fragments and skewed amplicon abundance. This is a common problem in multi-template PCRs; even small differences in amplification efficiency between templates can lead to vastly different template ratios after exponential amplification. This bias can depend on the relative difference between amplicon lengths as well as the overall amplicon size.

For genotyping samples with large differences in amplicon length (such as insertions or deletions), the reported allele frequencies may not reflect the true population. If quantitative results are necessary, contact Plasmidsaurus' Custom Sequencing services.

Rerun Policy

You are welcome to submit a rerun request for any failed sample through your Order Info page or via the support@plasmidsaurus.com email address. We will evaluate whether your sample quality and quantity permits rerunning the sample (and we may also ask you to provide a reference sequence).

Sample quality checks may require any of the following: