The right answers to frequently asked questions
Find the answers to all our products and services by clicking the links below.
General Questions regarding Sample Submission, Ordering & Sample Shipment & Results can be found here:
When should I use the Custom Long-Read Amplicon Sequencing service instead of the ONT Lite Clonal Amplicon Sequencing service?
Custom Long-Read Amplicon Sequencing dedicates an entire flow cell to your amplicons. As a result, this method is more time-consuming and costly compared to ONT Lite Clonal Amplicon Sequencing service, but it is highly suitable in the following cases:
- When you need additional services like 16S Microbiome Analysis, Unique Sequence Analysis, Variant Analysis, Sequence Cleaning, Host Removal, Pod5 raw data delivery
- When your linear DNA sample is not clonal but instead contains a mix of different molecules.
- When full-length, end-to-end reads are required, without any fragmentation.
- When full-length assemblies without missing terminal nucleotides are required.
- When a higher number of sequencing reads is needed beyond the typical output of the ONT Lite Clonal Amplicon Sequencing service.
- When you need to obtain all raw reads generated from your sample.
How should I purify my amplicons?
We recommend purifying your amplicons with commercially available kits based on DNA-binding beads or columns or enzymatic cleanup. DO NOT ship any primers with your samples or mixed into your samples.
What types of samples can be submitted for amplicon sequencing?
Submit purified PCR products with a size range of 500 bp to 25 kb. The amplicons should ideally produce a single band on a gel for optimal results.
I have mixed amplicons in a single sample that I want to sequence. Can I get the unique sequences from this mixture, and how can your service help with this?
Yes, you can send in a single sample containing mixed amplicons for sequencing. Our Unique Sequence Analysis service is specifically designed to handle such samples. By using advanced sequencing and bioinformatics techniques, we can accurately separate and identify each unique sequence, ensuring you get comprehensive and reliable results from your mixed amplicon sample.
How accurate are the sequencing results?
According to Oxford Nanopore's specifications for the chemistry and flow cells we currently use, the consensus accuracy is typically greater than 99.99%
How about addressing the errors or positions with low confidence that I discovered in a homopolymer region or at a Dam or Dcm methylation site?
The prevalent error modes in Oxford Nanopore sequencing include deletions within homopolymer stretches, errors occurring at the central position of the Dcm methylation site CCTGG or CCAGG, and errors at the Dam methylation site GATC.
So please handle these regions with special care.
What data & file types will I receive?
- Raw Nanopore amplicon data (FASTQ)
- Raw Nanopore sequencing data (POD5) available on request (additional costs)
- Customizable data analysis packages are available in our ordering form
- Variant analysis
- Variant (SNPs & InDels) Calls (TSV)
- Analysis Report (HTML)
- 16S Microbiome Analysis
- OTU Quantification Tables (TSV)
- Analysis Report (HTML)
- Unique Sequence Analysis
- Unique Sequences (FASTA)
- Clustering quantification table (TSV)
- Analysis Report (HTML)
- Consensus Sequence Analysis
- Consensus Sequence (FASTA)
- Variant Calls (TSV)
- Analysis Report (HTML)
What type of analysis is provided for Custom Long Read Amplicons?
Various analyses can be selected during ordering, including 16S microbiome, consensus sequence analysis, variant detection and unique sequence identification, are available depending on your project's specifics. If you require a special analysis, please reach out to your sales representative.