Key Specs
NGS short reads, 99 kr./Gb (€ 15, min. 10 Gb)
Nanopore long reads, 6,999 kr./Flowcell (€ 959)
Turnaround time: 25-30 workdays on average, max. 40 workdays. Holidays include Denmark, UK, and China.
Workflow
Sample Requirements
For DNA samples submitted, Customer is required to ensure the Quality Control (QC) data at the time of sample submission meets the minimum requirements concerning the total amount, concentration, and quality of the DNA submitted. For microbial culture or direct environmental samples submitted, Customer is obligated to ensure that the sample submitted is non-pathogenic to humans and does not contain any substance that may pose a health risk to the personnel working with the samples in our laboratory.
DNA sample
Total quantity ≥ 400 ng
Concentration ≥ 20 ng/µL; Volume ≥ 20 µL
Purity: A260/280 = 1.8 – 2.0
Slight deviations are usually fine. QC-failed samples will be considered high-risk ones of which data quality is not guaranteed and will be fully charged even if sequencing fails.
Notes:
(1) The final concentration of each sample will be measured using QUBIT or an equivalent fluorometer after the samples are received. If the samples do not pass QC, We will notify the Customer, and Customer shall either notify us to cancel the failed samples or re-send the samples concerned within ten workdays. Customer can request that the “QC-failed samples” be sequenced, in which case we will prepare the library and sequence the “QC-failed samples” without bearing any responsibility for the data quality. Customer should notify whether plasmid, chondriosome, chloroplast, and other non-bacterial-origin DNA exist in the samples submitted. Customer should notice that, If the DNA originates from a mixture of multiple microbial species, the quality of final assembly and genome annotation will be compromised significantly, and we will not attempt to separate all assemblies into individual species by any means.
(2) An aliquot of DNA sample may be stored temporarily in case of a failed library preparation. All DNA samples will be discarded within 3 months after the project is completed.
Microbial pure culture sample
Agar plates:
colonies should be easily visible and cover > 20% of the surface area on a standard-size petri dish (⌀ 90mm); the agar should not dry out and maintain a good texture for easy collection of cells with a standard sterile loop used in microbiology laboratories.
Pellets:
Bacterial pellets should be placed in a 1.5 mL or 2 mL centrifuge tube. The size of the pellet shall not be less than that generated by centrifuging 2 mL of E. coli cultures grown in LB overnight at 37 °C, typically covering a round area of ⌀3-5 mm.
Notes:
Customer should pay close attention to contamination on agar plates or in bacterial pellets. Customer must ensure each sample submitted is a single pure culture without being mixed with other species. In any case, we will continue the workflow and sequence the genomic DNA extracted from the samples submitted. We will not accept the complaint on the culture’s purity issue filed by Customer based on the genome sequence data generated in this project.
Environmental sample
The direct submission of soil, lake water, filters, swabs, plant or animal materials, any items or objects colonized by microbes, and other types of environmental samples that contain microbial inhabitants for genomic investigation at our laboratory is subject to individual agreement by email or in other written form between Customer and us concerning the sample type, quantity, and quality before the commence of this agreement.
Deliverables
The project will only commence when we receive the last batch of qualified samples. When a project is successfully completed, we will provide the following items as the standard deliverables:
DNA QC report;
Recommended 10-200 Gb sequence data per sample depending on sample complexity, with base quality of Q20 >95% in fastq format;
Contamination and low-quality reads from raw reads are removed before delivery;
Basic bioinformatics analysis results, including reads QC, reads assembly into contigs, gene prediction and annotation, taxonomic classification, and biodiversity stats. The computational work is performed by a machine-supervised automation pipeline w/o manual corrections depending on the Customer’s orders;
Project final report.
Notes:
1) National holidays include those in Denmark, UK, and China; 2) the following sample deliverables may contain items exclusively included in the Pro or Premium service package.
Pricing & Turnaround Time
For short reads-based sequencing projects, the flat rate is 99 kr. (€ 15) per Gb of data and 79 kr. (€11) for ≥ 20 samples sent in a batch. A minimum of 10 Gb of data is typically required for a sample. Please contact us if customers would like to negotiate on this threshold. The genome will be sequenced on a short reads platform from either Illumina or MGI.
For long-read sequencing with Nanopore technologies, the rate is 6,999 kr. per flowcell per sample. A minimum of one Minion flowcell is required for each sample. If ≥ 10 samples are sent in a batch or ≥ 10 flowcells are used, the rate is 5,999 kr.
The project turnaround time is counted from when DNA is ready for sequencing library prep. It is estimated to be 25-30 workdays on average, at most 40 workdays. If the project is not finished within 40 workdays, we will charge no fees related to this project to Customer and refund the prepayment, if any. Please be aware that we are an international team; thus, holidays include Denmark, UK, and China. It is for the contract reason that we set this relatively safe time frame. In most cases, customers will receive the results and final reports within a month, which is our service target.
Add-on Services
The Pro Bioinformatics Package (1,699 kr.; € 229) include all Pro services listed on the workflow. The Premium Bioinformatics Package (9,199 kr.; € 1,299) is highly customer-tailored with the highest level of services provided to address any significant challenges facing customers in their applications; details upon inquiry.
Payment Term
All payments set forth on the invoice are due within 30 days from the date of the invoice. We may impose a service charge on past due amounts between one and one-half percent (1.5%) and three percent (3%) per month, not to exceed the maximum amount permitted by law. Upon the Customer’s request, we will provide a statement within ten days.
The service agreement should be governed by and construed in accordance with the law in Denmark.
Service Contract
References
Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., & Henrissat, B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research, 37(suppl_1), D233-D238.
Wood, D. E., & Salzberg, S. L. (2014). Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology, 15(3), 1-12.
Li, D., Liu, C. M., Luo, R., Sadakane, K., & Lam, T. W. (2015). MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 31(10), 1674-1676.
Mikheenko, A., Saveliev, V., & Gurevich, A. (2016). MetaQUAST: evaluation of metagenome assemblies. Bioinformatics, 32(7), 1088-1090.
Chen, L., Zheng, D., Liu, B., Yang, J., & Jin, Q. (2016). VFDB 2016: hierarchical and refined dataset for big data analysis—10 years on. Nucleic Acids Research, 44(D1), D694-D697.
Huerta-Cepas, J., Szklarczyk, D., Forslund, K., Cook, H., Heller, D., Walter, M. C., … & Bork, P. (2016). eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Research, 44(D1), D286-D293.
Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y., & Morishima, K. (2017). KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Research, 45(D1), D353-D361.
Bolyen, E., Rideout, J. R., Dillon, M. R., Bokulich, N. A., Abnet, C. C., Al-Ghalith, G. A., … & Caporaso, J. G. (2019). Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology, 37(8), 852-857.
Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20(1), 1-13.
Zeng, Y., Chen, X., Madsen, A. M., Zervas, A., Nielsen, T. K., Andrei, A. S., … & Hansen, L. H. (2020). Potential rhodopsin-and bacteriochlorophyll-based dual phototrophy in a high Arctic glacier. mBio, 11(6), e02641-20.
Bioinformatics
Tools Frequently Used:
Bracken, Megahit, CD-HIT, Quast, emapper, humann2, kneaddata, Trimmomatic, bowtie2, diamond, salmon, Kraken, EMBOSS, FastQC, qiime2, Prodigal, SortMeRNA, SAMtools, Prokka, RefineM, CheckM, metabat2, metawrap, etc.
Here is the collection of scripts and Linux commands.
FAQ
Q: How much sequence data should I request for my project?
The short answer is that it entirely depends on your sample type and project objectives. The amount of data needed varies a lot among projects, ranging from a few Gb to hundreds of Gb per sample. For a quick scan of functional potential in a simple microbial community like tap water or cloud microbiota, 5 Gb may be already enough. But for complex communities like soil or gut, it is common to sequence >100 Gb per sample, so you have the best chance to reconstruct the genomes of unculturable members at a high accuracy and coverage level.
Q: What is the benefit of having both Nanopore long reads and NGS short reads for a hybrid de-novo assembly?
Long reads can improve the completeness and accuracy of genome bins or so-called metagenome-assembled genomes (MAGs) generated with bioinformatic tools. You will also have a better chance to reconstruct the genomes of rare members (<1% abundance) in the microbial communities. We expect that such a hybrid sequencing strategy will become more popular in the near future than present since long-read sequencing cost keeps decreasing.
Q: Do you accept DNA or soil samples preserved in a freezer for years?
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.
Q: Do you use any proprietary software for bioinformatic analysis?
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 publications. We aim to make all protocols available on our websites and keep them updated over time by following the latest literature.
