Life Science/Next Generation Sequencing/

Microbiome Analysis

Incredible 16S, 18S and ITS Microbiome Analysis Promotion

 

Flexible Voucher Option: Save your Funds and Budgets!

Buy now and perform this or any other project within 12 months.

 

“All in One Microbiome” Packages (16S, 18S and ITS): High Throughput Amplicon Generation, Sequencing and Analysis

Package A: 192 samples as batch only 32 € per sample and region

Package B: 96 samples as batch only 50 € per sample and region
Package C: 48 samples as batch only 75 € per sample and region
Package D: 10 samples as batch only 199 € per sample (limited to V4 region)

 

Other samples amounts are also possible (no shared runs)!

Microbiome flyer Download MiSeq Sequencing Order Form

Chose one option for amplicon sequencing or combine bacterial and fungal microbiome analysis

(1 amplicon = 1 sample):

 

Bacterial and Archaea microbiome analysis:
16S, V4 region, Primer combination: 515F – 806bR* or
16S, V4-V5 region, Primer combination: 515F – 909R* (=924R*) or

16S, V3-V4 region, Primer combination: 341f – 806bR****

! Please note that for 16S regions the quality of read 2 is significant lower (15-20%) than read 1. This is not an sequencing or quality error!

 

or Fungal microbiome analysis:
ITS, ITS 1 region, Primer combination: ITS1F – ITS2** (Note: ITS1F primer is 38 bp upstream of ITS1 from White et al., 1990)

 

or Eukaryotes (microbial) analysis:

V9 region, Primer combination: Illumina_Euk_1391f – Illumina_EukBr_1510r, blocking primer optional***; V9 region samples could not be combined with ITS od 16S samples and need to be sequenced in a seperate MiSeq run.

 

Send us your gDNA samples and make use of our “All in One” Service:
• Quality control
• Single step amplicon generation with reduced bias
• Double indexing, quality check, quantification, normalization and pooling of amplicons
• Illumina MiSeq sequencing package A-C (16S + ITS): exclusive 2 x 300 nt paired-end sequencing with V3 chemistry

• Illumina MiSeq sequencing package A-C (18S): exclusive 2 x 250 nt paired-end sequencing with V2 chemistry full flow cell

• Illumina MiSeq sequencing package D (16S + ITS + 18S): exclusive 2 x 250 nt paired-end sequencing with V2 chemistry nano flow cell
• Output for package A-C (16S + ITS): 15-30 M reads (including up to 25 % PhiX to balance the composition of bases)*

• Output for package A-C (18S): 10-20 M reads (including up to 25 % PhiX to balance the composition of bases)*

• Output for package D (16S + ITS + 18S): 750 K reads (including up to 25 % PhiX to balance the composition of bases)*
• De-multiplexing of reads
• Free 16S metagenomics analysis (Illumina App)
• Data delivery via FTP server

*Please note that for 16S the output is lower than Illumina specifies for standard chemistry V2 and V3 runs.

 

Applications:
• Environmental Metagenomics (soil, water, air, biofilms, complex organic communities)
• Human or Animal Microbiome (skin, stool, gut, blood, swab)

• Monitoring of animal health (e.g. aternatively or subblementerily to FELASA-Test)
• Sterility Monitoring
• Detection of Contamination
• Monitoring of Biogas Plant
• Biosafety Monitoring
• Food Quality
• Clinical Samples

 

Sequencing of your ready to load 16S/ITS/18S libraries:

• Illumina MiSeq sequencing: 2 x 300 nt paired-end sequencing with V3 chemistry or 2 x 250 nt paired-end sequencing with V2 chemistry
• V3 chemistry output is 15-30 M reads (including up to 25 % PhiX to balance the composition of bases)

• V2 chemistry output 10-20 M reads (including up to 25 % PhiX to balance the composition of bases)
• De-multiplexing of reads
• Free 16S metagenomics analysis (Illumina App)
• Data delivery via FTP server

 

Only 2600 €

 

Primer sequence references:

*Apprill A, McNally S, Parsons R, Weber L. 2015. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 75:129–137. Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol. 2016;18: 1403–1414.

*Parada, A. E., Needham, D. M., & Fuhrman, J. A. (2016). Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology, 18(5), 1403–1414. https://doi.org/10.1111/1462-2920.13023

*Walters, W., Hyde, E. R., Berg-Lyons, D., Ackermann, G., Humphrey, G., Parada, A., … Knight, R. (2016). Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. mSystems, 1(1), e00009-15. https://doi.org/10.1128/mSystems.00009-15

**White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322 In: PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, NY. Gardes, M., and T. D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol. Ecol. 2: 113-118.

***Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W., & Huse, S. M. (2009). A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes. PLOS ONE, 4(7), e6372. Retrieved from https://doi.org/10.1371/journal.pone.0006372

**** Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M., et al. . (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, 1–11. 10.1093/nar/gks808

****Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing. Bourtzis K, ed. PLoS ONE. 2014;9(8):e105592. doi:10.1371/journal.pone.0105592.

 

see also https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html

 

Simply order by e-mail: microbiome@starseq.com

 

You have no time/staff for routine preparation of gDNA from your collected samples? As an additional service we offer also the purification of gDNA from various starting material. Please contact us if you are interested in further service options or other gene regions and we will find YOUR optimal solution!
info@starseq.com

Bioinformatic analysis of 16S/18S/ITS sequences (QIIME 2)

 

The bioinformatics analysis pipeline consists of the following steps including quality control, pre-processing of reads, taxonomy identification and visualization, calculation of alpha and beta-diversity metrics and test for differential abundance of features between sample categories.

  • Raw reads are de-multiplexed and quality checked by FastQC¹.
  • Primers are trimmed using the tool cutadapt².
  • Paired-end reads are joined by the tool VSEARCH³.
  • Low-quality reads are removed.
  • Reads are corrected and Amplicon Sequence Variants (ASVs) are obtained by the deblur workflow4 and rare ASVs are filtered out.
  • 16S/18S: A multiple sequence alignment and a phylogenetic tree is generated using the tools MAFFT5 and FastTree6.
  • Alpha-diversity rarefaction curves are generated for categories and each individual sample.
  • Taxonomy is assigned to ASVs using a naive Bayes approach of the scikit-learn Python library7 database. Interactive stacked bar-charts of the taxonomic abundances of each category and each sample are generated.
  • Alpha and beta diversity metrics are calculated after normalization by rarefaction. Alpha-diversity Shannon metric boxplot are generated comparing different categories.
  • A test for difference in the relative abundance of features between categories is carried out by ANCOM10 if ordered.

Notes:
Taxonimical assignment is performed down to species level if possible. Due to reasons like sample quality, genus and family diversity and database composition the assignment might only be possible down to genus or family.
Since the ITS region shows more variation than 16S/18S a reliable multiple sequence alignment is not possible11. Therefore the alignment and the tree is not generated for this region and analyses based on diversity matrices (e. g. UniFrac12) leading to erroneous results are not carried out.

 

  1. Andrews S. (2010) FastQC: a quality-control tool for high-throughput sequence data. Babraham Institute, Cambridge, United Kingdom.
  2. Martin, M. (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. journal, 17(1), pp. 10-12.
  3. Rognes T., Flouri T., Nichols B., Quince C., Mahé F. (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. doi: 10.7717/peerj.2584
  4. Amir A., McDonald D., Navas-Molina J.A., Kopylova E., Morton J.T., Zech Xu Z., Kightley E.P., Thompson L.R., Hyde E.R., Gonzalez A., Knight R. (2017) Deblur rapidly resolves single-nucleotide community sequence patterns. mSystems 2:e00191-16. https://doi.org/10.1128/mSystems.00191-16.
  5. Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids research, 30(14), 3059–3066.
  6. Price, M. N., Dehal, P. S., & Arkin, A. P. (2009). FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Molecular biology and evolution, 26(7), 1641–1650. doi:10.1093/molbev/msp077
  7. Bokulich, N. A., Kaehler, B. D., Rideout, J. R., Dillon, M., Bolyen, E., Knight, R., … Gregory Caporaso, J. (2018). Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome, 6(1), 90. doi:10.1186/s40168-018-0470-z
  8. Nilsson, R. H., Larsson, K. H., Taylor, A., Bengtsson-Palme, J., Jeppesen, T. S., Schigel, D., … Abarenkov, K. (2018). The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic acids research, 47(D1), D259–D264. doi:10.1093/nar/gky1022
  9. Mandal, S., Van Treuren, W., White, R. A., Eggesbø, M., Knight, R., & Peddada, S. D. (2015). Analysis of composition of microbiomes: a novel method for studying microbial composition. Microbial ecology in health and disease, 26, 27663. doi:10.3402/mehd.v26.27663
  10. Halwachs B., Madhusudhan N., Krause R., Nilsson R.H., Moissl-Eichinger C., Högenauer C., Thallinger G.G., Gorkiewicz G. Critical Issues in Mycobiota Analysis. Microbiol. 2017;8:180.
  11. Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R. 2011 UniFrac: an effective distance metric for microbial community comparison. ISME J. 2011 Feb; 5(2):169-72.

Microbial DNA preparation service from various starting material:

 

StarSEQ has long term and advanced knowledge in extracting DNA from a wide range of starting material. We adjust varied DNA extraction protocols, kits and bead mill grinding to meet the individual demands of any project. We are using the most advanced tissue homogenizer from Bertin (Precellys Evolution and Minilys) for optimal adapted sample preparation.

Our experience covers almost all kind of genetic material:

 

• All kind of tissues and blood

• Swabs from skin and other surfaces

• Fecal samples

• Saliva samples

• Soil and sluge

• Sediments
• Plant tissues, roots, seeds or leafs
• Fungi tissues and spores

• Water and liquids

• Air filter

• Biofilms

• Nutriments

 

Starting from 18€/sample

Sequencing of your ready to load 16S/ITS/18S libraries:

 

• Illumina MiSeq sequencing: 2 x 300 nt paired-end sequencing with V3 chemistry
• Output 20-30 M reads (low cluster density including 25 % PhiX to balance the composition of bases)
• De-multiplexing of reads
• Free 16S metagenomics analysis (Illumina App)
• Data delivery via FTP server

 

Only 2600 €

Download MiSeq Sequencing Order Form
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