?

FAQ

Most commonly asked questions and answers. We keep things simple and straightforward. What you see is what you get. No hidden cost. No price discrimination. We see trust as fundamental to our business.

Share This Post

Table of Contents

General

We accept most types of samples as long as they do not pose any health risk to our staff working in the laboratory. In other words, we do not work with samples that potentially contain human pathogens, f.x. fresh saliva swabs from COVID-19 patients. But if such samples are pre-treated with chemical preservation solutions that kill viable entities before being sent to us, we will accept them.

This can vary significantly among different projects. Thus, we avoid proposing a general requirement for all types of samples regarding the sample quantity. We will have a detailed discussion during a project meeting or a general inquiry. As a rule of thumb, 1/4 to half of a 1.5 mL centrifuge tube of soils or sediments is sufficient.  But much more quantity will be required for samples with low microbial cell abundance.

Yes, but please don’t hold high expectations. Between 5-10% off is a possible offer we can provide for a large project. Generally, we adopt a transparent pricing policy without discrimination for any customer, whether a big pharma or a startup. So you are clear upfront before contacting us. We always strive to keep the cost for our customers low so that everyone can benefit from technological advances in microbial genomics. Please contact us for a quote on a specific project.

No. We only use open-source programs to ensure the reproducibility of our results. All protocols are publicly available on our website, or customers will be referred to original scientific papers. We aim to make all protocols available on our websites and keep them updated over time by following the latest literature.

We are an international team. The national holidays in Denmark, the UK, and China are considered when counting workdays. Please follow the link to see the holidays for the whole year.

DNA Extraction

We usually regard 1 ng/µL DNA  as the minimum acceptable concentration for DNA isolation. We will handle these cases individually. If it is because of human errors that happen during our processing of the samples, we will charge no fees for these samples. We will repeat the whole process on the same sample in such cases. If it still does not work out well, we will conclude that the submitted samples are unsuitable for DNA isolation using standard protocols, and we will charge a small reagent fee of 30 kr./sample. We will do our best to avoid such a situation by carefully examining the samples and consulting with clients for more details about samples before commencing a project.

Both column-based and magnetic bead-based DNA extraction methods are widely used for isolating and purifying DNA from various sample types. Each method has its advantages and drawbacks, and the choice between them often depends on factors such as the specific application, desired yield, and purity requirements.

Column-based DNA extraction uses silica spin columns to bind and purify DNA, while magnetic bead-based extraction relies on DNA binding to coated magnetic beads. Both methods provide high yield and purity. Column-based extraction is suitable for small to medium sample sizes but can be challenging to scale up or automate. Magnetic bead-based extraction is well-suited for automation, high-throughput applications, and larger or challenging samples but can be more expensive. The choice depends on factors such as yield, purity, scalability, automation, and cost.

Microbial Genome Sequencing

Unfortunately, you can’t. No matter how deep you sequence the genome with short reads methods, you won’t generate a closed genome. Short reads cannot span the repetitive regions longer than the length of the reads themselves. Repetitive sequences are widespread in microbial genomes due to genome evolution. Thus, there are always gaps in short reads-assembled genomes. Most of the gaps are located in the regions containing transposons, phage-related sequences, or genes of recombinases or integrases.

Maybe in the not-far future but not present. Although Nanopore reads’ accuracy is improving, it is still way below that of short reads generated from Illumina or MGI sequencers. New developments put Nanopore reads into the Q30 category but only on a limited fraction of reads at a small scale and relying on expensive chemistries. The cost-effectiveness of short reads is unmatched. Short NGS reads are often used to polish erroneous Nanopore long reads. Combining Nanopore’s long reads and NGS’s short reads is the best strategy to close a small genome with very high accuracy.

It is not guaranteed. This workflow was designed using a small bacterial or archaeal genome as a reference. For a larger genome, the sequencing depth should be accordingly higher. Usually, we recommend 50-100x long reads and 200-500x short reads to generate a good hybrid assembly. The more complex a genome is, the more reads are desired. Our experience shows it’s much harder to close a yeast/fungal/micro-eukaryote’s genome than a bacterial genome.

Microbial Community Profiling

The 16S rRNA gene is approximately 1,500 base pairs long and contains both highly conserved and variable regions, consisting of 9 variable regions (V1-V9) and these regions can be paired in different ways to create amplicons for sequencing. Some of the most commonly used variable regions and their comparisons are:

 

V1-V3 region:

  • Length: ~500 base pairs.
  • Taxonomic resolution: Provides good resolution at the genus level.
  • Primer choice: Well-established primers are available for this region.

 

V3-V4 region:

  • Length: ~460 base pairs.
  • Taxonomic resolution: Offers good resolution at the genus level and is often considered the “gold standard” for 16S rRNA gene amplicon sequencing.
  • Primer choice: Well-established primers, such as 341F/805R, are available and provide good coverage across bacterial and archaeal taxa.

 

V4 region:

  • Length: ~250-290 base pairs.
  • Taxonomic resolution: Provides excellent resolution at the genus level and sometimes at the species level.
  • Primer choice: Well-established primers, such as 515F/806R, are available and provide good coverage across bacterial and archaeal taxa.
  • Compatibility: The short amplicon length is compatible with popular sequencing platforms like Illumina, which is one of the reasons why the V4 region is often preferred for microbial community profiling.

 

V4-V5 region:

  • Length: ~380 base pairs.
  • Taxonomic resolution: Offers good resolution at the genus level.
  • Primer choice: Primers targeting the V4-V5 region are available, but they may not provide as comprehensive coverage as those for the V3-V4 or V4 regions.

 

The choice of which variable region(s) to target for 16S amplicon sequencing depends on factors such as the organisms of interest, the desired taxonomic resolution, sequencing platform constraints, and the availability of well-established primers. Additionally, some researchers may choose to sequence multiple variable regions to achieve a more comprehensive understanding of microbial community structure. Ultimately, the selection of variable regions should be tailored to your specific research context and objectives.

The 18S rRNA gene is more conserved and longer than the 16S rRNA gene, with a length of approximately 1,800 base pairs. While there are no standard designations for the variable regions in the 18S rRNA gene as there are for the 16S rRNA gene, several regions are commonly targeted for amplicon sequencing:

 

V4 region:

  • Length: ~400-500 base pairs.
  • Taxonomic resolution: Provides good resolution at the genus or family level for various eukaryotic taxa.
  • Primer choice: Well-established primers are available for this region, such as TAReuk454FWD1/TAReukREV3.
  • Compatibility: The amplicon length is compatible with popular sequencing platforms like Illumina.

 

V5 region:

  • Length: ~300-400 base pairs.
  • Taxonomic resolution: Provides moderate resolution at the genus or family level for various eukaryotic taxa.
  • Primer choice: Primers targeting the V5 region are available, but they may not provide as comprehensive coverage or taxonomic resolution as primers for the V4 or V9 regions.

 

V7 region:

  • Length: ~300-400 base pairs.
  • Taxonomic resolution: Offers moderate resolution at the genus or family level for various eukaryotic taxa.
  • Primer choice: Primers targeting the V7 region are available, but like the V5 region, they may not provide as comprehensive coverage or taxonomic resolution as primers for the V4 or V9 regions.

 

V9 region:

  • Length: ~150-200 base pairs.
  • Taxonomic resolution: Offers good resolution at the genus or family level for various eukaryotic taxa.
  • Primer choice: Well-established primers are available for this region, such as Euk1391f/EukBr.
  • Compatibility: The short amplicon length is well-suited for high-throughput sequencing platforms like Illumina.

 

The choice of which region(s) to target for 18S amplicon sequencing depends on factors such as the organisms of interest, the desired taxonomic resolution, sequencing platform constraints, and the availability of well-established primers. Keep in mind that the 18S rRNA gene is more conserved than the 16S rRNA gene, and in some cases, researchers might target other markers like the internal transcribed spacer (ITS) regions for fungal diversity or 28S rRNA and COI (cytochrome c oxidase subunit I) for specific eukaryotic taxa. As always, the selection of target regions should be tailored to your specific research context and objectives.

The short answer is that it entirely depends on your sample type and project objectives. The number of tags generated from short reads-based amplicon sequencing varies among projects, commonly chosen between 30k, 50k, and 100k. For most sample types, 30k tags work pretty well. You may consider 50k or 100k tags for extremely complex communities like soils or human/animal guts. But for PacBio long reads-based profiling, the cost increases rapidly if you go beyond the standard 10k CCS reads. Please contact us for further information.

Both methods produce highly accurate reads. With long PacBio reads, you get significantly longer 16S/18S/ITS fragments than short reads from an Illumina or MGI sequencer. Thus, a higher resolution of resolving microbial community structure can be achieved with PacBio reads, often down to the strain level. But PacBio sequencing is relatively expensive, so the sequencing depth will usually be sacrificed, limiting its ability to address highly complex microbial communities. Regarding turnaround time, a project involving PacBio sequencing takes roughly one more week to complete than that only with NGS.

The choice between using the internal transcribed spacer (ITS) regions ITS1 and ITS2 for profiling fungal community structure depends on the goals of your study and the specific organisms you are interested in. Both regions are widely used as molecular markers for fungal identification and diversity analysis, and they have their respective advantages and disadvantages.

 

ITS1:

  • Higher variability: ITS1 is usually more variable than ITS2, which can make it a better choice for distinguishing closely related species or strains.
  • Less prone to co-amplification: ITS1 is less likely to co-amplify plant DNA in environmental samples, which can be an advantage when studying fungal communities in plant-associated environments.

 

ITS2:

  • More conserved secondary structure: ITS2 has a more conserved secondary structure, which can make it easier to design primers and align sequences.
  • Better reference database coverage: ITS2 often has better coverage in reference databases, which can improve the accuracy and confidence of species identification.

 

Some researchers choose to use both ITS regions to achieve a more comprehensive understanding of fungal community structure. It is important to consider the organisms you are targeting, the goals of your study, and the availability of primers and reference databases when making your decision. Ultimately, the choice between ITS1 and ITS2 will depend on your specific research context and objectives.

The lengths of the internal transcribed spacer (ITS) regions ITS1 and ITS2 can vary between different fungal species and even within a single species due to the presence of insertions, deletions, or other sequence variations. Generally, the ITS1 region is considered to be more variable in length than the ITS2 region. Here are the approximate length ranges for each region:

 

ITS1:

  • The length of the ITS1 region typically ranges from 100 to 350 base pairs (bp), although in some species, it can extend to over 1000 bp.

 

ITS2:

  • The ITS2 region is generally shorter and more conserved in length, usually ranging from 140 to 300 bp.

 

This variation in length can impact the choice between ITS1 and ITS2 for profiling fungal community structure. Longer and more variable regions, like ITS1, can provide higher resolution for distinguishing closely related species or strains. However, this variability can also make it more challenging to design universal primers or align sequences for phylogenetic analysis.

In contrast, ITS2’s more conserved length and secondary structure can simplify primer design and sequence alignment. Ultimately, the choice between ITS1 and ITS2 for your study should consider these factors along with the specific organisms of interest and your research goals.

Environmental Metagenomics

Yes, we do. DNA is amazingly stable when stored in TE buffer under a freezing temperature. Frequent thaw-freeze may break DNA double strands to some level but generally has a minor effect on sequencing output and data quality. We also accept frozen soil samples and provide professional services to extract high-quality DNA from these samples. Please be aware that sequencing results from a fresh soil sample can differ a lot from the same sample put in a freezer for years since microbial communities change slowly, even under freezing temperatures.

MetaSPAdes and MEGAHIT are both widely used metagenome assembly tools. While they employ different algorithms and strategies to assemble metagenomic data, their overall goals are to reconstruct the original genomes of microorganisms in a metagenomic sample. Below are some differences between the two assemblers:

 

MetaSPAdes:

  • MetaSPAdes is an extension of the SPAdes assembler designed specifically for metagenomics.
  • It uses multiple k-mer sizes and iteratively refines the assembly.
  • MetaSPAdes is known for producing high-quality assemblies and has shown better performance than MEGAHIT in some studies when it comes to contig accuracy and genome reconstruction.
  • However, MetaSPAdes tends to be more computationally demanding (in terms of RAM usage and running time) than MEGAHIT, especially for large and complex datasets.

 

MEGAHIT:

  • MEGAHIT is a standalone metagenomic assembler that employs succinct de Bruijn graphs and iterative assembly strategies.
  • It is specifically designed to handle large metagenomic datasets with limited computational resources.
  • MEGAHIT is typically faster and more memory-efficient than MetaSPAdes, which can be advantageous for large-scale metagenomic projects or when computing resources are limited.
  • While MEGAHIT may produce slightly lower quality assemblies compared to MetaSPAdes, it still generates reliable results.

 

In summary, MetaSPAdes tends to produce higher quality assemblies but is more computationally intensive, while MEGAHIT is faster and more memory-efficient but may yield slightly lower quality results. The choice between the two assemblers largely depends on your specific needs, dataset size, and available computational resources. It is also worth noting that assembly quality can vary between datasets, so it may be beneficial to try both assemblers and compare their results using benchmarking tools like MetaQUAST or BUSCO.

Emergent Sequencing

We highly recommend taking this approach, as it will help reduce the time required to complete the project. We are also eager to collaborate with our clients in the lab to achieve the best results in the shortest possible time.

We adopt a transparent pricing policy for all customers, whether you are a big pharma, a public institution, or a small startup. This also applies to emergent sequencing services. Please refer to the current rates shown above for consumables and sequencer running costs. The Customers are encouraged to do their own calculations. We will refund the difference overcharged if there are any deviations between your calculations and ours. In essence, we only charge labour costs and a one-off setup fee.

This is guaranteed, as we state in our service targets, because we have our own bioinformatics servers in-house with automated pipelines, which are constantly monitored remotely by a small but highly capable and efficient data scientist team. The only thing is that we need to adjust the priority of existing projects to serve emergency projects better.

Data Analysis Package

As a general policy, we do not disclose the locations of our servers and genome data scientist team. But we can confirm that all our resources are distributed within these three countries: Denmark, UK, and China. In some cases, we may use AWS, Azure, or Alibaba Cloud to perform certain parts of the pipelines. If you are concerned that your data may leave the EU territory, please seek other bioinformatics service providers, as we cannot guarantee that.

The easiest way is to upload the data to a shared Dropbox folder we create for each project. Customers can also use other online big file transfer services or even mail hard disks to us. Once the project is done, we will return the disks by regular mail.

Yes, you will. Many of our customers are academic researchers who need a detailed description of the whole bioinformatic pipeline for future publications. We have a ready-for-publication version of all our protocols including parameter settings for each command or program. This will be included in a project’s final report. We will make all methods and protocols available online at some point.

The figures will be in an uneditable PNG or JPG format with a maximum resolution of 300 dpi for the Basic package. All figures will be in both SVG and PNG/JPG formats for the Pro or Premium package. The SVG files can be edited with Adobe Illustrator or Inkscape to generate publication-quality figures.

Subscribe To Our Newsletter

Get updates and learn from the best

More To Explore
Admin

Holidays 2024

This is a summary of official holidays of 2024 in Denmark, the UK, and China. We use this calendar to count the number of workdays spent on a project.

Read More »
Technical documents

Microbial Genome Sequencing

Sequencing a microbial genome has never been as easy and affordable as now. We provide a complete sequencing package with the basic bioinformatics analysis included. Our professional services allow you to be hustle-free and focus on your critical tasks.

Read More »
Technical documents

Microbial Community Profiling

Microbial life dominates our planet Earth in terms of quantity and biodiversity. The very first step to understanding them is to know who they are. This can be addressed via high-throughput amplicon sequencing of a few conservative biomarker genes.

Read More »

live a life as light as microbes

Do You Want To share your project or recieve a quote?

Contact Us