Genotyping Analysis (Beta)
Characterize allelic composition and frequency at a single locus.
Genotype with ease and speed
Accurate characterization of allelic composition and frequency at defined loci is critical, yet traditional workflows remain limited. Sanger sequencing lacks the ability to phase variants, while both Sanger and Illumina are constrained by amplicon length.
Plasmidsaurus redefines the genotyping workflow. We’ve eliminated the bottlenecks: no more PCR cleanup, no more library prep, and no sequencing primers. Whether your amplicons are short or massive, we deliver high-quality readouts of allelic composition and frequency in just 1–2 days. Send your samples today; get your insights tomorrow.
Note: Genotyping Analysis detects alleles present at >10% frequency and substitutions or small insertions/deletions under 200 bp in size. For more complex samples or to assess larger insertions/deletions, please consider our premium PCR or custom sequencing services.
Get clear answers, fast
Confidently characterize the sequence and frequency of each allele in your sample across amplicons of any length. Understand sequence changes at the nucleotide and amino acid level with data that you can’t get from Sanger or Illumina.
Say goodbye to data wrangling
Eliminate complex and tedious analysis pipelines. See visualizations comparing aligned alleles and get sequences and allelic frequencies in 1-2 days.
Key applications
Cell line, model organism, or germplasm QC
Confirm your cell line or model system contains a desired allele after gene editing or after receiving samples from an external source.
Locus-specific genotyping
Profile variants at defined loci to characterize their sequence. For example:
- You have a new mouse model showing a disease phenotype. You want to understand the variation at a specific gene.
- You discovered a new strawberry variant. You want to know if there is allelic variation at a specific locus.
Characterize your genetic crosses
Genotype specific loci in progeny or hybrids.
Biomarker-based stratification
Use locus-specific variants to group cell lines and assess genotype–phenotype relationships.
Phase variants
Understand if variants are present on the same strand of DNA (cis) or different strands (trans).
Screen transgenics
Identify founders with a target genotype.
“I've got two distinct indels, so it's a knockout! This tool is awesome! I'm definitely going to recommend this service to friends in my department!”
“Many times our CRISPR-edited mouse founders have multiple variants, and Sanger just can’t deconvolute the linked mutations. This new allele calling pipeline does the work for us. It clearly connects mutations in cis, gives us frequency of alleles, and saves us time digging through noisy raw reads. A huge improvement!”
Data deliverables & bioinformatics
Allele specific files
Virtual gel
Per base data, prior to allele calling
Feature annotations and reference alignments
Sequencing statistics including read length and coverage
Raw reads
Product specs & service levels
- Long read sequencing with ONT
- Optimized for linear, double-stranded DNA
- Add sample cleanup for $5
| Service Level | Size | Target Read Depth/Sample* | Minimum Volume | Concentration* | Cost | Target Turnaround Time |
|---|---|---|---|---|---|---|
| Standard | 100 bp - 25 kb | Up to 3,000 reads | 10 μL | 1 ng/μL per 100 bp | $30 | 1-2 days |
* Sequencing is based on the molarity of your DNA insert, therefore the required concentration (ng/uL) will vary depending on your insert size.
Ready to sequence?
Put your DNA in a tube and drop it off. Click below for requirements and suggestion for optimal results.
Related products
Amplicon
Need to verify cloning and gene editing with a specific region? Try Amplicon Sequencing.
AAV
Sequencing AAV? Check out our AAV sequencing service for an ultra-fast service that returns comprehensive data with minimal sample input.
Whole Plasmid
Our Whole Plasmid Sequencing service with ZeroPrep lets you skip culture and minipreps and get straight to your results faster.
FAQs
We sequence each sample with Oxford Nanopore long reads to very high depth before generating reads using the latest basecalling and polishing software. Then we cluster these reads into phased alleles.
- We construct an amplification-free long-read sequencing library using the newest v14 library prep chemistry
- We sequence the library with a primer-free protocol using the most accurate R10.4.1 flow cells.
- We remove noise, phase alleles, and report frequencies only for alleles >10%. Frequencies are adjusted so that reported alleles total 100%.
- If we detect a sequence at a frequency below 10%, the tool assumes it is likely due to PCR or sequencing error and assigns those reads to a “major” allele (one that has >10% allele frequency) that is most similar in sequence.
- Example: When a major allele is identified at 48% frequency and a closely related sequence is identified at 2%, the lower-frequency sequence is merged with the major allele. The final reported frequency of the major allele is therefore 50%.
Our pipeline phases variants into assembled sequences called alleles. If you provide a reference sequence, we align these alleles to your reference.
In some samples, your provided reference sequence may be the same as one of the observed alleles (for example one allele is WT and one allele is a mutant). If this is the case, we will denote this allele with a label eg Allele 1 (Your reference sequence name).
There may be cases when the provided reference is not one of the observed alleles, for example, in cases when all alleles are mutants. In these cases, we will only show your provided reference in the nucleotide and amino acid comparison view.
Finally, if you choose not to provide a reference sequence, we will align all sequences to the sequence with the highest frequency (denoted as Allele 1).
We have tested the genotyping analysis at a range of different allelic ratios for SNPs and small indels. In general we find a strong correlation between experimentally prepared ratios and pipeline-predictions (R2 > .98). If you notice significant discrepancies between our analysis and your expected results, please reach out to us at support@plasmidsaurus.com. To learn more about our data deliverables, see the Genotyping Analysis product page.
If your sample includes amplicons that differ significantly in length, for example due a large insertion or deletion on one allele, the pipeline will perform separate analyses on each amplicon and thus report allele frequencies for each amplicon separately. For example, in the case of a simple heterozygous locus with a large indel, you will see two amplicon maps, one for each sized amplicon, each with one allele at 100% allelic frequency.