Troubleshooting guide
progress bar
progress bar

Frequently asked questions
Contact us
Subscribe to newsletter
w10.0.14 | c1.25.03.1
PROD | u7.5.14
  Quick Order  0
Ecom-Admin
  • Markets
  • Products
  • Company
  • Contact
  • EN
  • Oligonucleotide Synthesis
  • Gene Synthesis & Molecular Biology
  • Sanger Sequencing
  • Next Generation Sequencing
  • Genotyping & Gene Expression

Further Information:

>> EVOcard

>> Product FAQs

>> SALE %

Application Oligos

  • qPCR Probes
  • Ultimate Precision Probes
  • PCR Primer
  • SeqPrimer
  • Cloning Oligos
  • NGSgrade Oligos 2.0
  • NGSgrade Oligos
  • NGS UDI Primer Sets
>> Show more

Custom Oligos

  • Custom DNA Oligos
  • Salt-Free Oligos
  • Large Scale Oligos
  • Custom RNA Oligos
  • Special Requests
>> Show more

Oligo Tools

  • Oligo Analysis Tool
  • PCR Primer Design Tool
  • Sequencing Primer Design Tool
  • qPCR Assay Design Tool
  • siRNA Design Tool
  • Prepaid Oligo Coupons
  • Excel Order Forms
>> Show more

Benefit from more than 25 years of experience in oligonucleotide synthesis!

>> Show all products

Gene Synthesis

  • Standard Genes
  • Express Gene
  • Gene Synthesis Project
  • GENEius
>> Show more

GeneStrands

  • GeneStrands
  • Express GeneStrands

Molecular Biology Services

  • Plasmid Preparation
  • CRISPR/Cas9
>> Show more

Optimise your research and save time with high quality gene synthesis and molecular biology services.

>> Show all products

Sequencing Services

  • Mix2Seq Service
  • LightRun Services
  • TubeSeq Supreme
  • PlateSeq Supreme
  • Direct Colony Sequencing
  • Whole Plasmid Sequencing
  • Primer Walking Service
>> Show more

Prepaid Products

  • Mix2Seq Kits
  • LightRun Barcodes
  • Barcode Labels & Coupons
  • PlateSeq Kits
  • PlateSeq Kit Colony
  • ONT Prepaid Coupons
>> Show more

Additional Services

  • Free Barcode Labels
  • Sequencing Accessories
  • Sequencing Primers
  • Sample Shipment
>> Show more

Hiqh quality Sanger sequencing with highest flexibility for every sample type.

>> Show all products

Top 5 Products

  • INVIEW Resequencing
  • INVIEW Microbiome
  • INVIEW Transcriptome
  • INVIEW Metagenome
  • Amplicon sequencing
  • Bioinformatic services
  • Genome sequencing
>> Show more

Spotlight

  • INVIEW Exome
  • INVIEW Virus
  • INVIEW CRISPR Check
  • NGS Prepaid Coupons
  • Oncology Solutions
  • OLINK Explore 3072
  • Clinical Research Exome
  • Bacteria Hybrid Assembly
>> Show more

Oxford Nanopore Products

  • Whole Plasmid Sequencing
  • Linear / Clonal Amplicons
  • Bacterial Genome Sequencing
  • 16S Full-Length Microbiome
  • Complete ONT Portfolio
>> Show more

NGS from experts - ISO-certified, fully automated and easy to order online.

>> Show all products

Applied Genomic Services

  • DNA Barcoding
  • Cell Line Authentication
  • Mycoplasmacheck
  • Fragment Length Analysis
>> Show more

Genotyping services

  • SNP Genotyping
  • Copy Number Variation
  • Mikrosatellites/ STR/FLA/ IDAA
>> Show more

Gene Expression Services

  • Transcriptome analysis
  • Expression Arrays
  • Target Gene Expression
>> Show more

Cell line authentication, Mycoplasmacheck, Fragment length analysis & tailored projects.

>> Show all products
  • Pharma / Biotech
  • Agrigenomics
  • Consumer Genomics
  • Food & Environment
  • Diagnostic Kit Producer
  • Research / Biotech

Further Information:

>> Quality

>> Events

Synthesis Products

  • Industrial-grade NGS Oligos
  • Ultimate Precision Probes
  • Special Oligo Requests
  • Large Scale Oligos
  • Gene Synthesis
  • GeneStrands
  • Plasmid Preparation

Sequencing Services

  • Amplicon Sequencing
  • Exome Sequencing
  • Transcriptome Sequencing
  • Primer Walking Service

Favorite Content

  • NGS – Disease Diagnostics
  • Cancer Ecology
  • Personalised Cancer Therapy
  • Revolutionising human-like-protein production
  • The Microbiome Of Cancer
  • All about biomarker discovery

Our genomics solutions support you along your drug development chain of small and large molecules and in precision medicine.

>> Show all products

Plant Breeder

  • DNA marker discovery
  • Marker-assisted selection
  • GRAS-Di®
  • Microbiome and metagenomics

Animal Breeder

  • DNA Marker discovery
  • Pathogen screening
  • Parentage testing
  • Genomic selection
  • Marker-assisted selection

Favorite Content

  • Microarrays Accelerate Blue Biotechnology
  • How To Do NGS 50% Faster
  • NGS Portfolio

Benefit from our range of tailored, high throughput genotyping solutions to help you achieve your goals faster, for less.

>> Show all products

Sequencing Services

  • Genotyping
  • Epigenome Profiling
  • Microbiome Analysis
  • Shotgun Sequencing
  • Whole Genome Sequencing

Additional Services

  • Sample Collection Kits
  • Shipping
  • DNA Extraction
  • Laboratory Service
  • Biobanking

Favorite Content

  • Population Genetics
  • The End of Gene Doping
  • Home Genomics Testing
  • IVDR Compliance

Welcome to your full-service laboratory. Benefit from our end-to end solutions for sample collection kit logistics and genomic solutions.

>> Show all products

Food Testing

  • Food Authenticity
  • Meat Traceability
  • Pathogen Traceability
  • Cannabis and Hemp Testing

Environmental Testing

  • Non-targeted detection of organisms / species
  • Targeted detection of organisms / species
  • eDNA Tracker

Favorite Content

  • Determine the Source of Meat
  • Pine Nuts – Why Testing For Edibility Matters

DNA-based solutions to improve and support your analysis, monitoring and traceability across your value chain.

>> Show all products

Synthesis Products

  • Large Scale Oligos
  • Special Requests in Tubes
  • Special Requests in Plates

Quality Assurance

  • GLP
  • ISO 17025
  • ISO 13485

Favorite Content

  • Oligonucleotides For Diagnostics
  • The Future of RNA Applications

Our scalable and high-quality oligonucleotides synthesis offer makes us an ideal partner for your industry applications.

>> Show all products

Favorite Services

  • Custom DNA Oligos
  • TubeSeq Services Sanger
  • INVIEW Microbiome NGS
  • NGS Services
  • GeneStrands

Express Services

  • TubeSeq NightXpress
  • Mix2Seq NightXpress
  • Express Genes
  • Express GeneStrands

Favorite Content

  • Eurofins Genomics Goes Green
  • GENEius – Codon Usage Optimisation
  • 5 tips to speed up your qPCR

For your research project in academic, governmental and industrial environment we have the right genomic service.

>> Show all products

Corporate Information

  • About us
  • Career
  • Events
  • Press Releases
  • Collaborations
  • Blog
  • Newsletter
  • Quality Assurance
  • Lab closure times

Help

  • Video Tutorials
  • Ordering
  • Payment
  • Shipping & Delivery
  • Downloads
  • Data & Privacy
  • Material and Methods

Contact us

Eurofins Genomics
Germany GmbH
Anzinger Str. 7a
85560 Ebersberg
Germany

  • phone +49 7531 816068
  • e-mail support

Get in touch with us!

You need support or advice with your order? You don't find the perfect product or you like to get consultation regarding your results?

>> Get in touch

Find your product

Find your product

  • Sequencing Primer
  • PCR Primer
  • Mycoplasmacheck
  • SALE %
  • TubeSeq

Login to Eurofins

Please login with your email address and password!

>> Login

New at Eurofins Genomics?

Register a new account at Eurofins Genomics!

>> Register
Impersonating: Max Mustermann

Administration

  • Ecom Admin
  • Stop Impersonating

Welcome

Julian Schlossmacher

Login / Register

 
Email
Email can not be empty
Password:
Password can not be empty
Forgot password?
Create Account
No SSL
>> Logout >> My profile

Contact

Dr. Melvin Siliakus

+31 629 39 25 66 >> Send message

Account

  • Overview
  • Orders
  • Quotes
  • EVOcards
  • Preferences
  • DropBoxes
  • Sanger Kits & Barcodes
  • NGS Barcodes & Coupons
  • Sanger Samples & Primers
  • Oligo Coupons

Language

model-logo

New Website Navigation explained

>> Close
6

Last added items

0 Item(s)

Go to Cart

>> Go to cart X
 

Quick Order

X

Industrial-grade Products & Services

What are industrial-grade products? <<click here >>

Oligonucleotide Synthesis

  • Ultimate Precision Probes
  • NGSgrade Oligos in Tubes
  • NGSgrade Oligos in Plates
  • Request for Oligos in Tubes
  • Request for Oligos in Plates
  • Large Scale Oligos

Gene Synthesis & Molecular Biology

  • Gene Synthesis
  • GeneStrands
  • Express GeneStrands
  • Plasmid Preparation
  • Cloning Service

Next Generation Sequencing

  • Genome (Re-)Sequencing
  • Transcriptome Sequencing
  • Microbiome profiling
  • Exome Sequencing
  • Clinical Research Exome
  • Oncoprofiling
  • Liquid Biopsy Sample Sequencing
  • Ready-to-Load
  • Virus Sequencing
  • Amplicon Sequencing
  • Metagenome
  • ONT products
  • NGS Barcodes & UPS labels
  • Replacement samples
  • NGS Additional Services

Genotyping & Genexpression

  • Cell Line Authentication
  • Fragment Length Analysis
  • Mycoplasmacheck Barcodes
  • Cell Line Barcodes

Oligonucleotide Synthesis

Primers & Probes for qPCR Applications

  • PCR Primer in Tubes
  • PCR Primer NightXpress
  • PCR Primer in Plates
  • LocNA Primer
  • Dual Labeled Probes
  • MGB Probes
  • LocNA Probe
  • Molecular Beacons
  • LightCycler Probes
  • Probe based qPCR Assay
  • Ultimate Precision Probes

Oligos for Next Generation Sequencing

  • NGSgrade Oligos 2.0 Tubes
  • NGSgrade Oligos 2.0 Plate
  • NGSgrade Oligos in Tubes
  • NGS Oligos for NovaSeq
  • NGS Oligos for MiSeq
  • NGS UDI Primer Sets

Primers for Sanger Sequencing

  • SeqPrimer in Tubes
  • SeqPrimer NightXpress
  • SeqPrimer in Plates
  • Standard Primer

Oligos for Cloning Applications

  • Cloning Oligos
  • EXTREmer Oligos

Custom Oligos

  • Custom DNA Oligos in Tube
  • SaltFree Oligo NightXpress
  • Custom DNA Oligos in Plates
  • Custom RNA Oligos
  • O-Methyl-RNA / Chimerics
  • RNA qPCR Probes
  • siMAX siRNA
  • Special Oligos in Tubes

Oligo Tools

  • Oligo Analysis Tool
  • PCR Primer Design
  • SeqPrimer Design
  • qPCR Assay Design
  • siRNA Assay Design
  • Prepaid Oligo Coupons

Gene Synthesis & Molecular Biology

Synthetic genes

  • Gene Synthesis
  • Combinatorial libraries
  • Gene Synthesis Projects

GeneStrands

  • GeneStrands
  • Express GeneStrands

Molecular Biology Services

  • Plasmid Preparation
  • Site Directed Mutagenesis
  • DNA Cloning Service

Sanger Sequencing

Sanger Sequencing Services

  • TubeSeq Supreme
  • PlateSeq Supreme
  • Direct Colony Sequencing
  • Ready2Load Plate
  • Ready2Load Tube
  • Whole Plasmid Sequencing Tubes
  • Whole Plasmid Sequencing Plate
  • TubeSeq NightXpress
  • Primer Walking Service

Prepaid Products for Sanger Sequencing

  • Prepaid Barcodes & Coupons
  • Mix2Seq Kits
  • PlateSeq Kits
  • LightRun Barcodes
  • ONT Coupons

Additional Services

  • Tube & Plate Barcode Labels
  • Sequencing Accessories
  • Sample Shipment
  • Sequencing Primer Design

Next Generation Sequencing

Genome Sequencing

  • INVIEW Resequencing
  • NGSelect DNA
  • Whole Genome Sequencing
  • Bact. Whole Genome Seq (ONT)

Transcriptome Sequencing

  • INVIEW Transcriptome
  • NGSelect RNA

Metagenome / Microbiome

  • INVIEW Microbiome
  • INVIEW Metagenome

Exome Sequencing & Oncology Solutions

  • INVIEW Human Exome
  • Liquid Biopsy Samples
  • INVIEW Oncoprofiling
  • Clinical Research Exome

CRISPR & Prepaid NGS Coupons

  • INVIEW CRISPR Check
  • Redeem NGS Coupons
  • Order NGS Coupons

VIRUS

  • INVIEW Virus

Plasmid Sequencing

  • INVIEW Plasmid Verification
  • Whole Plasmid Sequencing (ONT) Tubes
  • Whole Plasmid Sequencing (ONT) Plate

Amplicon sequencing & Ready-to-Load

  • NGSelect Amplicon
  • NGSelect Ready-to-Load
  • Clonal Amplicons (ONT)

Oxford Nanopore projects (WGS, Amplicons, 16S)

  • ONT projects

ONT Lite

  • ONT Lite Whole Plasmid Sequencing
  • ONT Lite Clonal Amplicon Sequencing
  • ONT Lite Bacterial Genome Sequencing
  • ONT Lite Assembly Review

Additional Services

  • NGS Barcodes & UPS labels
  • NGS Additional Services
  • Sample Submission Guidelines
  • Prepaid NGS Coupons
  • Replacement samples
  • Request for Information

Genotyping & Gene Expression

Mycoplasmacheck

  • Mycoplasmacheck Barcodes
  • Mycoplasmacheck results

CLA & FLA

  • Cell line authentication Service (CLA)
  • Fragment length Analysis (FLA)
  • Cell line authentication barcodes

Others

  • Genotyping request form

EVOcard

Order / Refill EVOcard

Login / Register

 
Email
Email can not be empty
Password:
Password can not be empty
Forgot password?
Create Account
No SSL

 

  • Order Menu

    EVOcards

    • Order / Refill EVOcard

    Oligonucleotides & siRNA

    • (q)PCR Primer in Tubes
    • (q)PCR Primer in Plates
    • (q)PCR Primer NightXpress
    • SeqPrimer in Tubes
    • SeqPrimer in Plates
    • SeqPrimer NightXpress
    • Custom DNA Oligos in Tubes
    • Custom DNA Oligos in Plates
    • SaltFree Oligo NightXpress
    • NGSgrade Oligos in Tubes
    • Standard Primer
    • Standard Primer NightXpress
    • LocNA Primer
    • LocNA Probes
    • Dual Labeled Probes
    • MGB Probes
    • Probe based qPCR Assay
    • Cloning Oligos in Tubes
    • EXTREmer Oligos
    • Nano-Scale Plate Oligos
    • NGS UDI Primer Sets
    • Custom RNA Oligos
    • RNA qPCR Probes
    • siMAX siRNA
    • Large Scale Oligos
    • Special Oligo Requests
    • More...

    Custom DNA Sequencing

    • Mix2Seq Kits
    • LightRun Barcodes
    • Sequencing Primers
    • TubeSeq Service
    • SupremeRun Tube
    • Tube & Plate Barcode Labels
    • TubeSeq Labels & Coupons
    • SupremeRun Barcodes
    • Sequencing Accessories
    • PlateSeq Kits
    • SupremeRun 96
    • Primer Walking
    • PlateSeq Service
    • SupremeRun | Multiprimer
    • Sequencing Projects
    • Direct Colony Sequencing
    • Ready2Load Plate
    • More...

    Next Generation Sequencing

    • INVIEW Microbiome 3.0
    • NGSelect DNA
    • Barcode & UPS Labels
    • INVIEW Transcriptome
    • NGSelect RNA
    • Replacement samples
    • INVIEW Resequencing
    • NGSelect Amplicon
    • Sample Submission
    • INVIEW Human Exome
    • Redeem NGS Coupons
    • Request for Information
    • INVIEW CRISPR Check
    • SARS-CoV-2 RNA-Seq
    • More...
    • INVIEW Virus

    Gene Synthesis & Molecular Biology

    • New gene wizard
    • Plasmid Preparation
    • GeneStrands
    • Express Genes
    • DNA Cloning Service
    • Express GeneStrands
    • Gene Synthesis Project
    • Site Directed Mutagenesis
    • Combinatorial libraries
    • Synthetic genes
    • Corona Control Plasmids

    Genotyping & Gene Expression

    • Mycoplasmacheck
    • Cell Line Authentication 2.0
    • Fragment Length Analysis
    • Request for Information

0 Item(s)

Go to Cart

  • Other Languages
  • DNA & RNA
    Oligonucleotides
    • >

      Optimised Application Oligos

    • >
      Ultimate Precision Probes
    • >
      PCR Primers
    • >
      SeqPrimer
    • >
      Cloning Oligo
    • >
      EXTREmers
    • >
      NGSgrade Oligos
    • >

      qPCR Probes

    • >
      NGSgrade Oligos 2
    • >
      Custom DNA & RNA Oligos
    • >
      Custom DNA Oligos
    • >
      Large Scale Oligos
    • >
      Custom RNA Oligos
    • >
      siMAX siRNA
    • >
      Special Requests
    • >
      Salt Free Oligos
  • Custom DNA Sequencing
    • >

      Eurofins Services

    • >
      Mix2Seq
    • >
      Mix2Seq Kits
    • >
      TubeSeq Services
    • >
      TubeSeq Labels & Coupons
    • >
      PlateSeq Service
    • >
      PlateSeq Kits
    • >
      Direct Colony Sequencing
    • >
      Whole Plasmid Sequencing
    • >

      LightRun Services

    • >
      LightRun Barcodes
    • >
      GATC Services
    • >

      Additional Services

    • >
      Tube & Plate Barcode Labels
    • >
      Sequencing Primers
    • >
      Sequencing Accessories
    • >
      Sample Shipment
    • >
      Free Sample Pick-Up
    • >
      Shipping Options
  • Next Generation Sequencing
    • >

      NGS Built For You

    • >
      INVIEW Microbiome
    • >
      INVIEW Transcriptome
    • >
      INVIEW Exome
    • >
      INVIEW Metagenome
    • >
      INVIEW CRISPR Check
    • >
      INVIEW Virus
    • >
      NGS prepaid solutions
    • >
      INVIEW Plasmid Verification
    • >

      NGS Build Your Own

    • >
      NGSelect DNA
    • >
      NGSelect RNA
    • >
      NGSelect Amplicons
    • >
      NGSelect Ready2Load
    • >
      Human Whole Genome Sequencing
    • >
      WGS Select
    • >
      Bacteria Whole Genome Sequencing
    • >
      Amplicon sequencing
    • >
      Amplicon sequencing - full service
    • >

      Applications

  • Gene Synthesis / Molecular Biology
    • >

      Gene Synthesis

    • >
      Standard Genes
    • >
      Express Genes
    • >
      Complex Genes
    • >
      GeneStrands
    • >
      Express GeneStrands
    • >
      Combinatorial Libaries
    • >
      GENEius
      • >

        Molecular Biology Services

      • >
        Plasmid Preparation
      • >
        Site Directed Mutagenesis
      • >

        Applications

      • >
        CRISPR_Cas9
    • Genotyping & Gene Expression
      • >

        Service Platforms

      • >
        Illumina Array Platforms
      • >
        Affymetrix Array Platforms
      • >
        Fluidigm BioMark & EP1
      • >
        Roche LightCycler
      • >
        Sanger & NGS Sequencing
      • >

        Genotyping Services

      • >
        SNP Genotyping
      • >
        Microsatellites / STR / FLA / IDAA
      • >
        Sequencing Based Genotyping
      • >
        Genotyping by PCR
      • >
        Genome Wide Association
      • >
        Human Identification
      • >
        Copy Number Variation
      • >

        Gene Expression Services

      • >
        Transcriptome Analysis
      • >
        Expression Arrays
      • >
        Target Gene Expression
      • >
        miRNA / Small RNAs Analysis
      • >

        Applied Genomics Services

      • >
        Residual DNA Analysis
      • >
        DNA Barcoding
      • >
        Cell Line Authentication
      • >
        Mycoplasmacheck

    Troubleshooting & result interpretation

    • You are here:
    • Custom DNA Sequencing >
    • Eurofins Services >
    • Whole Plasmid Sequencing >
    • WPS data interpretation

     

     

    Result interpretation guide for whole plasmid sequencing

     

    In our whole plasmid sequencing service we not only deliver a high-quality assembled and polished sequence, but also provide an informative HTML report. In case you need assistance interpreting the quality of your data, please find detailed descriptions below, along with examples of good and bad quality results. Additionally, we offer tips and tricks to ensure that you achieve only high-quality results.

     

    Oxford nanopore technologies (ONT) sequencing

     

    In ONT nanopore sequencing, the input material directly influences the output. If a flow cell is loaded with small fragment DNA, the resulting reads will be of a corresponding size. Conversely, if high-molecular weight DNA is loaded, longer reads will be obtained. Overall, all molecules included in the sample will be sequenced and the relative read counts of various molecular species will generally align with the actual proportions of those species present in the sample. There is one caveat though: small fragments are sequenced in a higher frequency than longer ones.
    Also be aware that agarose gels don’t have a very high sensitivity, only DNA fragments in a high enough concentration can be visualized. There might be more going on at a lower level in the background of the sample (you might see a smear on the gel).
    Nanopore sequencing by ONT does not require primers and typically involves sequencing the entire plasmid molecule with read lengths that span its entirety. Therefore, all molecules present in the received sample, including degraded plasmids or background genomic DNA, are sequenced.

     

    Sample requirements:

     

    Our sample requirements are:

    Size category

    Length

    Concentration

    Min. Volume

    Regular

    2.5 – 25 kbp

    30 ng/ µl

    20 µl

    Large

    25 – 125 kbp

    50 ng/ µl

    30 µl

    XL

    125 – 300 kbp

    50 ng/ µl

    50 µl

     

    The required DNA concentration according to our specifications are between 30 and 50 ng/µl. We highly recommend fluorometric concentration measurements (e.g. Qubit) instead of photometric ones (e.g. Nanodrop), because of their significantly higher accuracy for double-stranded DNA. Photometric measurements frequently overestimate the samples’ DNA concentration. We often receive plasmids from customers who still measure their concentration with photometric measurements, which is the most common reason for failed attempts of plasmid sequencing. We will attempt to sequence your sample even when your sample doesn’t fulfill your requirements. We cannot guarantee success, but unfortunately, we must charge for our sequencing attempt .

    As our service is optimized for plasmid clonal population of molecules, we recommend controlling the quality of a sample (i.e. a single gel band), preferably as a linearized plasmid, on a gel or with a Bioanalyzer/Fragment Analyzer (however watch out for biological concatemers, see below). If your samples failed to be sequenced, please consider performing a new plasmid preparation to rule out contamination. Additionally, performing a size selection on a gel could be a good procedure to remove contaminating degraded DNA.

     

     

    Accuracy of plasmid sequencing results

     

    According to the specifications provided by Oxford Nanopore for the chemistry and flowcells used in our current plasmid sequencing, the raw read accuracy exceeds 99%. In general, higher coverage, which refers to having more reads available for consensus building, tends to enhance the accuracy of the results.

    Nevertheless, we also deliver a variant calling within the report, which detects positions in the final plasmid sequence with lower confidence.

    If you observe discrepancies between our consensus and your reference, it is possible that the plasmid construct you provided differs from your reference due to missing elements, mutations, or other factors. Such outcomes are commonly revealed through whole plasmid sequencing.

     

     

    Lower confidence bases

     

    Our consensus assembly process utilizes deep sequencing to achieve a high level of accuracy at the individual base level. However, Oxford Nanopore long read data encounters challenges in resolving certain common motifs. To tackle this issue, we polish the sequence to correct many of these problematic bases.

    In addition, we employ a strategy where we map your reads against a high-quality consensus assembly to identify lower confidence bases. During this process, we determine the frequency of each nucleotide at a specific position. In regions with high confidence bases, the majority of raw reads will contain the same assembled base. However, in areas that pose challenges, such as motifs like Dcm methylation sites (CC[A/T]GG) or long stretches of homopolymer bases, different nucleotides may be identified at the same position in the raw reads, despite the assembled base potentially being correct.

    If your assembly differs from your expectations, it is important to consider these factors.

     

     

    Errors or low confidence positions in homopolymer region or a Dcm / Dam methylation site

     

    The most common error modes for Oxford Nanopore are deletions in homopolymer strechtes, errors at the middle position of the Dcm methylation sites CCTGG or CCAGG and errors at the Dam methylation site GATC.

     

     

    Sequencing coverage of plasmids

     

    We cannot provide a specific level of coverage guarantee as the number of raw reads generated can significantly vary based on the quality of the sample. Typically, successful samples sent at the recommended concentration yield a substantial number of raw sequencing reads, ranging from high dozens to potentially hundreds or even thousands. The average coverage is indicated in the report, and a coverage of approximately 20x or higher suggests a highly accurate consensus.

     

     

    For more questions, please also visit our FAQs >>

     

    Data interpretation

     

    Read length histograms

     

    Prior to sequencing your plasmids, the library preparation workflow linearizes the circular DNA to obtain predominantly full-length sequence reads. In the result report we plot a read length histogram, which shows the read length from all DNA molecules present in the sample (and which can be sequenced). One dominant peak in the histogram indicates a clean plasmid preparation, usually with a sufficient concentration (see good examples below).

     

     

    Non-weighted vs. weighted histogram

     

    Distribution of read lengths from sequenced data is shown in the following histograms. Read length histograms can be used to assess the quality of sequencing data, as the distribution of read lengths can indicate extraction quality and fragmentation, the presence of contaminants, or biases in the sequencing process. They can also be used to determine the size of short plasmids, depending on the quality of the sample.

     

     

    Non-weighted histogram

     

    The first histogram displays the number of reads on the y-axis and the read length on the x-axis. Each bar in the histogram represents a range of read lengths, and the height of the bar indicates the total number of reads falling within that range.

     

     

    Weighted histogram

     

    The second histogram displays the number of sequenced bases (bp) on the y-axis and the read length on the x-axis. Instead of total number reads the height of the bars indicate the total number of bases (bp) falling within that range. This results in a weighted plot by the number of nucleotides per bin, as longer reads carry more weight in the histogram.

     

     

     

    Examples

     

    Example of good plasmid preparations

     

         

     

    One dominant peak indicates a clean monoclonal plasmid preparation, which usually yields good sequencing results (if enough coverage is achieved). Plasmid mixtures can result in erroneous assemblies, depending on sequence homologies within the sample.

     

    Please be aware that a single apparent peak in the histogram could represent multiple plasmids of the same size or multiple plasmids of varying lengths that happen to fall within the same bin. In the analysis pipeline, sequences that are highly similar are treated as variations of a single species, resulting in an attempt to generate a single consensus sequence (with potentially low confidence positions reported in the report).

     

     

    Example of plasmid preparations that meet the required criteria

    In certain instances, we encounter samples exhibiting a prominent peak alongside a significant presence of degraded DNA, including both genomic and plasmid fragments. In the majority of cases the dominant peak still yields a consensus sequence if the read coverage and accuracy meet the required thresholds.

     

             

     

     

    Sometimes the read length is divided into two different size bins of the histogram. This can be a result of:

    • defined bin boundaries split the dominant read length peak of one clean plasmid into two, this has no influence on our analysis
    • small variations in the plasmid (e.g. homopolymer regions of different sizes, small insertion and deletions (InDels)), which result in slightly different plasmid sizes around the bin boundaries
    • plasmid mixture of two plasmids of very similar size

     

    These variations in the plasmid prep can only be seen in the read length histogram but would not be visible in traditional Sanger sequencing.

     

    Example of multiple peaks

    You may observe multiple peaks in the read length histograms, this can have three reasons:

    • a sample including a mixture of plasmids with different sizes
    • Concatemers of a biological plasmid..
    • Unexpected side products of the propagation of the plasmid in the cloning strain that are a result of deletions, recombinations, or the above mentioned concatemers

     

    We frequently observe the presence of concatemers, which are not sequencing artifacts. Concatemers cannot be detected through Sanger sequencing, and they are not visible on gels of digested or linearized plasmids. Consequently, they may appear unfamiliar to those who are not accustomed to encountering them. However, running the sample uncut on a gel will reveal the dimer etc. band. Concatemers often form in vivo during growth in a recA+ strain. For a detailed description of this phenomenon see https://blog.addgene.org/plasmids-101-dimers-and-multimers

     

    As our service is optimized for plasmid clonal population of molecules, we recommend controlling the quality of a sample (i.e. a single gel band) on a gel or with a Bioanalyzer/Fragment Analyzer.

     

       

     

    Our pipeline is designed to only return the major peak and corresponding plasmid included in the sample (see weighted vs non-weighted above). If the above sample is a plasmid mixture only the plasmid with around 7kb would be reported.

     

    Resolving plasmid mixtures 

    Our plasmid service is designed for analyzing clonal populations of molecules. While it is possible to submit mixtures of molecular species, it comes with an inherent risk as we cannot predict the outcome of the analysis.

    • If your plasmid molecules are highly similar in length and sequence, with only a few nucleotide differences, the analysis pipeline will typically produce a single .fasta consensus file with low confidence positions at SNP/indel locations. You can refer to the report provided to identify these locations.
    • If your species exhibits sufficient distinctness, the pipeline will generate a .fasta consensus file for the most abundant species if the plasmid size is below <25kb.

     

    Degraded DNA or contamination with small fragments

    If your plasmid DNA is degraded during the preparation process, the resulting sequencing reads will predominantly consist of small fragments with no dominant peak, despite high read count. This can result in insufficient coverage and no plasmid consensus sequence can be generated. Another potential scenario is contamination with degraded host genomic DNA, which would have a similar effect. As our service is optimized for plasmid clonal population of molecules, we recommend controlling the quality of a sample (i.e. a single gel band), preferably as a linearized plasmid, on a gel or with a Bioanalyzer/Fragment Analyzer (however watch out for biological concatemers, see above).

     

       

     

     

    We do not offer re-sequencing or refunds for samples that fail, unless a technical issue is identified on our end. Samples not meeting the required DNA concentration, containing multiple plasmids, substantial host contamination, or plasmids with multiple large repetitive elements are more likely to fail in generating a consensus sequence. If your sample failed to be sequenced, please refer to our Sample requirement stated above, where you will find suggestions on how to improve your sample quality.

     

     

     

    Insufficient data

    Frequently, the read count is insufficient to differentiate individual peaks or generate a consensus. When the read count is too low, it is typically due to samples not being prepared at the required DNA concentration according to our specifications of 50 ng/µl. We highly recommend fluorometric concentration measurements (e.g. Qubit) instead of photometric ones (e.g. Nanodrop), because of their significantly higher accuracy for double-stranded DNA. Photometric measurements frequently overestimate the samples’ DNA concentration. We often receive plasmids from customers who still measure their concentration with photometric measurements, which is the most common reason for failed attempts of plasmid sequencing.

     

       

     

     

    Assembly failed/final plasmid size doesn’t fit

    Possible reasons are real-life biological plasmid concatemers (see above), which appear typically during propagation in recA+ strains via homologous recombination. If the concatemers are in a high proportion in the sample, they might be picked up. Concatemers cannot be seen on a gel with a digested/linearized plasmid, but you can run the supercoiled/uncut plasmid with a supercoiled ladder.

    Additionally, although the third gen ONT service sequences the plasmid with long reads in the plasmids’ size, in rare cases large highly repetitive plasmids might not be resolved correctly by the assembler. Finally, the sample quality might not be sufficient (a clean single plasmid prep has one dominant plasmid peak). Multiple peaks indicate plasmid mixtures, or other side products like insertions/deletions and/or recombination.

     

    Quality is important for us at Eurofins

    Our products and services are produced and performed under strict quality management and quality assurance systems.

    Find certificates here
    Production Site
    • Europe
    • America
    • India
    • Japan
    Eurofins Genomics
    • Terms & Conditions
    • Sitemap
    • Imprint
    • Privacy
    • Licenses
    • Cookie Settings
    Contact
    General Customer Support
    phone +49 7531 816068
    Toll free phone number for
    Europe: 00800 200 100 20
    support-eu@genomics.eurofinseu.com

    Eurofins Genomics Europe Shared Services GmbH
    Anzinger Str. 7a
    85560 Ebersberg
    Germany

    /p>

    2025 - Eurofins Genomics

    VEGA Beta