16S rRNA Sequencing Service

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16S rRNA gene sequencing, or 16S amplicon sequencing, is performed to determine the relative abundance of taxa in a bacterial community, and to compare between groups of interest. This level of analysis can help to address changes in the overall microbial profile over time, or between treatment groups. We have two 16s rRNA primer options available:

16S V4

16S V1-V3

We suggest speaking to a Microbiome Insights representative to better understand the pros and cons of each primer region to determine which one is best suited for your study.

Our 16S V4 and 16S V1-V3 workflow from DNA extraction, library preparation, sequencing and report generation have been accredited by the College of American Pathologists (CAP).

ITS2 rRNA Gene Sequencing Service

ITS2 rRNA gene sequencing, or ITS2 amplicon sequencing is performed to determine the relative abundance of taxa in a fungal community, and to compare between groups of interest.

See all our microbiome sequencing services.

DNA extraction and quality control

DNA is extracted from samples using the Qiagen MagAttract PowerSoil DNA KF Kit (formerly MO BIO PowerSoil DNA Kit) optimized for the Thermofisher KingFisher robot. This kit uses magnetic beads to specifically capture DNA while excluding organic inhibitors. In-house testing has shown that this kit has a good balance of DNA yield and quality, as demonstrated on a variety of environmental sample types.

DNA quantification and quality checks are done via Qubit.

Library preparation

We use the high-fidelity Phusion polymerase for amplification of 16s and ITS2 marker genes. If extractions contain some carry-over inhibition or a high concentration of DNA, typically we test 1:1 and 1:10 dilutions and run the PCR products on gels for verification. PCR is done with dual-barcoded primers (Kozich et al. 2014) targeting either the prokaryotic (16S V4, V1-V3) or fungal (ITS2) regions. Our barcoding strategy enables multiplexing up to 384 samples per run. PCR products are verified visually by running a representative subset of samples on a gel. Samples with failed PCRs (or spurious bands) are re-amplified by optimizing PCR conditions. The PCR reactions are cleaned up and normalized using the high-throughput SequalPrep 96-well Plate Kit. Samples are then pooled to make one library that is quantified accurately with the KAPA qPCR Library Quant kit.

16S & ITS2 Sequencing Platform

Library pools are sequenced on the Illumina MiSeq using the corresponding primer sets for either prokaryotic (16S V4, V1-V3) or fungal (ITS2) regions.

Certifications: All of our 16S & ITS2 amplicon sequencing services are performed in a College of American Pathologists (CAP) accredited laboratory.

Bioinformatics for 16S and ITS2

Our bioinformatic pipeline for 16S and ITS2 can accommodate different experimental designs. We have the capacity for microbial marker discovery and machine-learning-based modeling. Our main pipeline uses Mothur (Schloss, 2009) for processing amplicon data. We also have experience working with DADA2 and QIIME.

We deliver a fully comprehensive bioinformatics report with associated raw fastq files.

Standard bioinformatic reporting includes plots showing taxonomic composition, alpha diversity, beta diversity (ordination), differential abundance testing, and multi-variate analysis (permutational analysis of variance). This package also includes raw bioinformatics outputs (OTU table, taxonomic classification of each OTU), differential abundance results, and relative abundances of taxa at various taxonomic levels.

Learn more about our bioinformatics services.

Advanced bioinformatic reporting may include:

microbial epidemiology
multiple regression
partitioning sources of variation
meta-analysis
multi-omics integration
clustering algorithms

From point of initial contact to final bioinformatic report, I was thoroughly pleased with the amplicon sequencing and bioinformatics services provided by Microbiome Insights... the flexibility and willingness [of the team] to personalize the final bioinformatic report according to my needs was extremely helpful.

Irene Yang, PhD, RN
Assistant Professor at Emory University

Microbiome Insights

5

2018-11-22T17:07:58-08:00

Irene Yang, PhD, RN
Assistant Professor at Emory University

From point of initial contact to final bioinformatic report, I was thoroughly pleased with the amplicon sequencing and bioinformatics services provided by Microbiome Insights... the flexibility and willingness [of the team] to personalize the final bioinformatic report according to my needs was extremely helpful.

https://microbiomeinsights.com/testimonials/2566/

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Microbiome Insights

Frequenty Asked Questions

Sending Samples

Which amplicons do you run?

We can address taxonomic profiling for prokaryotic communities (16S V4, V1-V3 or V4_skin) and fungal communities (ITS2) on the Illumina Miseq using the version 3 chemistry.

Why do some labs select other primers?

Each primer has its pros and cons, so the primer you select depends on the aims of your particular study. 16s V1-V3 may produce better classification for skin microbiota; conversely the 16s V4 region is less suitable for skin taxa and classification. The 16s V4 region, however, produces an amplicon length that is more suitable for short-read sequencing, which reduces sequencing error.

How many reads do you get per sample?

For 16s and ITS2 rRNA gene sequencing the average number of reads per sample is close to 40-50K (pre-filtered). Most manuscripts tend to report pre-QC number of reads which will always be reduced after cleanup. The number of reads is driven by the number of multiplexed samples and the Miseq chemistry.

We recommend against over-sequencing as it inflates the number of spurious OTUs. The more reads that come out of the sequencer, the more likely those reads will create spurious OTUs.

We would generally not recommend omitting samples with low reads as there may be biological factors at play. Course-grained comparative analyses (e.g. beta diversity) are relatively robust to low reads.

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For information on sample collection, storage and transport please see our support section.

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