Chose one option for amplicon sequencing or combine bacterial and fungal microbiome analysis
(1 amplicon = 1 sample):
Single Step Bacterial and Archaea microbiome analysis:
16S, V4 region, Primer combination: 515F (5′-GTGYCAGCMGCCGCGGTAA-3′, recommended) optional (5′-GTGNCAGCMGCCGCGGTAA-3′, old version) – 806bR (5′-GGACTACNVGGGTWTCTAAT-3′)* or
16S, V4-V5 region, Primer combination: 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) – 926R (5′-CCGYCAATTYMTTRAGTTT-3′)* (recommemded) or
16S, V4-V5 region, Primer combination: 515F (5′-GTGNCAGCMGCCGCGGTA-3′) – 909R (=924R) (5′-CCCCGYCAATTCMTTTRAGT-3′)* (old version) or
16S, V3-V4 region, Primer combination: 341f (5′-CCTACGGGNGGCWGCAG-3′) – 806R (=786R) (5′-GACTACNVGGGTWTCTAATCC-3′)**** (recommended) or
16S, V3-V4 region, Primer combination: 341f (5′-CCTACGGGNBGCASCAG-3′) – 806bR (5′-GGACTACNVGGGTWTCTAAT-3′)**** (old version)
! 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 2-Step Bacterial and Archaea microbiome analysis:
16S, V1-V2 region, Primer combination 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) – 338R (5′-TGCTGCCTCCCGTAGGAGT-3′)
or Single Step Fungal (18S) microbiome analysis:
ITS, ITS 1 region, Primer combination: ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3′) – ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′)** (Note: ITS1F primer (Gardes and Bruns, 1993) is 38 bp upstream of ITS1 from White et al., 1990)
or 2-Step Fungal (18S) microbiome analysis:
ITS, ITS 1/2 region, Primer combination: ITS-u2 (5′-GAAYCATCGARTCTTTGAACGC-3′) – ITS-p4 (5′-CCGCTTAKTGATATGCTTAAA-3′)
ITS, ITS 2 region, Primer combination: ITS3F (5′-GCATCGATGAAGAACGCAGC-3′) – ITS4R (5′-TCCTCCGCTTATTGATATGC-3′)**
or 2-Step Eukaryotes (microbial) analysis:
V8-V9 region, Primer combination: 18S-1422f (5′-ataacaggtctgtgatgccct-3′) – 18S_1797R (5′-ccttcygcaggttcacctac-3′)*****
V9 region, Primer combination: Illumina_Euk_1391f (5′-GTACACACCGCCCGTC-3′) – Illumina_EukBr_1510r (5′-TGATCCTTCTGCAGGTTCACCTAC-3′), blocking primer optional***; V9 region samples could not be combined with ITS, V8-V9 or 16S samples and need to be sequenced in a seperate MiSeq run.
or 2-Step Barcoding:
cytochrome b, cytb1 (5′-ccatccaacatctcagcatgatgaaa-3′) – cytb2 (5′-gcccctcagaatgatatttgtcctca-3′)
cytochrome oxidase I, CO I E (5′-ccagagattagagggaatcagtg-3′) – CO 1 F (5′-cctgcaggaggaggagaycc-3′)
or 2-Step Custom Amplicons:
design of custom primers, request your special quotation: info@starseq.com
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
• 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, tissue, tumour)
• 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
• Data delivery via FTP server
Only 3300 €
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.
**Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes ‐ application to the identification of mycorrhizae and rusts. Molecular Ecology, 2(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.
***** Bradley, I. M., Pinto, A. J., & Guest, J. S. (2016). Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities. Applied and Environmental Microbiology, 82(19), 5878 LP-5891. https://doi.org/10.1128/AEM.01630-16
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
Nucl. Acids Res. 41 (D1): D590-D596.
