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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.

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Table of Contents

Key Specs
  • 16S/18S/ITS amplicon sequencing

  • 699 kr./sample (€ 99) with 30k short reads

  • 1,199 kr./sample (€ 159) with 10k CCS reads of near full-length 16S/18S/ITS from PacBio

  • Turnaround time: 25-30 workdays for short reads, 30-35 workdays for long reads, max. 40/50 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 ≥ 1.0 μg (short-insert library) or ≥ 5 μg (PCR-free library);

  • Concentration ≥ 20 ng/µL;

  • Purity: A260/280=1.8-2.0.

 

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

A project will only commence when we receive the last batch of qualified samples. If the project is successfully completed, we will provide the following items as the standard deliverables:

  • DNA QC report;

  • 30k/50k/100k raw tags per sample, depending on the Customer’s order, with an overall base quality of Q20 >95% in fastq format;

  • Basic bioinformatics analysis results, including reads QC, reads assembly, and OTU/ASV counts, including tentative phylum/genus/species classification information of each OTU/ASV in excel and fasta formats. The computational work will be performed by a machine-supervised automation pipeline integrating Qiime2 or USEARCH without manual corrections;

  • Project final report.

 

Notes

National holidays include those in Denmark, UK, and China.

Sample deliverables
  • DNA QC report
Pricing & Turnaround Time

For short reads-based 16S/18S/ITS amplicon sequencing projects, the flat rate is 699 kr. (€ 99) per sample and 599 kr. (€79) for ≥ 20 samples sent in a batch. 30k tags per sample are the standard output. This can be upgraded to 50k tags with a fee of 100 kr. and to 100k tags with 200 kr. The sequencing platform is either Illumina or MGI. 

For long reads-based 16S/18S/ITS  amplicon sequencing with PacBio, the flat rate is 1,199 kr. per sample. If ≥ 20 samples are sent in a batch, the rate is 999 kr. per sample. 10k CCS reads will be generated from each sample by circular consensus sequencing (CCS). 

The project turnaround time is counted from when DNA is ready for sequencing library prep. On average, it is estimated to be 25~30 workdays for short reads/30~35 workdays for long PacBio reads, at most 40/50 workdays. If the project is not finished within 40/50 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 timeframe. 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

Draft Agreement

References

Karp, P. D., Riley, M., Paley, S. M., & Pellegrini-Toole, A. (2002). The metacyc databaseNucleic acids research30(1), 59-61.

DeSantis, T. Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E. L., Keller, K., … & Andersen, G. L. (2006). Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and environmental microbiology72(7), 5069-5072.

Nepusz, G. C. A. T., & Csárdi, G. (2006). The igraph software package for complex network researchComplex Systems1695(5), 1-9.

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., … & Glöckner, F. O. (2012). The SILVA ribosomal RNA gene database project: improved data processing and web-based toolsNucleic acids research41(D1), D590-D596.

McMurdie, P. J., & Holmes, S. (2013). phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census dataPloS one8(4), e61217.

Dixon, P. (2003). VEGAN, a package of R functions for community ecology. Journal of Vegetation Science14(6), 927-930.

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 Research44(D1), D286-D293.

Wickham, H., Chang, W., & Wickham, M. H. (2016). Package ‘ggplot2’. Create elegant data visualisations using the grammar of graphics. Version2(1), 1-189.

Dinno, A., & Dinno, M. A. (2017). Package ‘dunn. test’CRAN Repos10, 1-7.

Yu, G., Smith, D. K., Zhu, H., Guan, Y., & Lam, T. T. Y. (2017). ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated dataMethods in Ecology and Evolution8(1), 28-36.

Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y., & Morishima, K. (2017). KEGG: new perspectives on genomes, pathways, diseases and drugsNucleic Acids Research45(D1), D353-D361.

Kolde, R., & Kolde, M. R. (2018). Package ‘pheatmap’R Package1.

Nilsson, R. H., Larsson, K. H., Taylor, A. F. S., Bengtsson-Palme, J., Jeppesen, T. S., Schigel, D., … & Abarenkov, K. (2019). The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classificationsNucleic acids research47(D1), D259-D264.

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 2Nature Biotechnology37(8), 852-857.

Douglas, GM, Maffei, VJ, Zaneveld, JR, Yurgel, SN, Brown, JR, Taylor, CM, … & Langille, MG (2020). PICRUSt2 for prediction of metagenome functions. Nature biotechnology , 38 (6), 685-688.

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 glaciermBio11(6), e02641-20.

Bioinformatics
Tools Frequently Used:

FastQC, Trimmomatic, QIIME2, Greengenes 16S database, Silva 18S database, UNITE ITS database, R package Phyloseq, R package ggtree, R package vegan, R package ggplot2, R package pheatmap, R package igraph, PICRUSt2, MetaCyc database, R package dunn.test, etc.

Here is the collection of scripts and Linux commands.

FAQ
Q: How many tags should I request for my project?

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.

Q: Which strategy should I choose between short NGS reads and long PacBio reads?

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.

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.

Q: Do you provide any discount for a large project involving hundreds of samples?
Yes, but please don’t expect high. 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 the technological advances in microbial genomics. Please contact us for the latest cost calculation on a specific project.
 
See more at mBioWorks FAQ.

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