Application Note
Metagenomic Service: 16S rDNA Analyses
Introduction
Amplicon deep-sequencing using next
generation sequencing (NGS) technologies has become an important and widely used tool to study the diversity in microbial communities. Next‐generation
sequencing technologies can generate
unprecedented amount of sequence
data enabling rapid and comprehensive
profiling of microbial communities by sequencing parts of the ribosomal DNA
(16S rDNA). However, the processing and
evaluation of next‐generation sequence
data is a challenge due to (i) the large
amount of data generated and (ii) the
technology‐specific sequencing patterns
which must be addressed properly dur-
ing downstream analysis. Microsynth offers a full service covering the entire process from DNA isolation, PCR amplification and sequencing up to bioinformatics analyses of the data. Final output
is a user-friendly report.
Workflow for Projects based on 16S rDNA
Project Input:
Option1:
Customer sends
environmental samples
Option 2: Customer sends
isolated DNA
Samples
(soil, tissue, feces, water, ...)
DNA Isolation
Selection of
Target Region
Design and Validation
of Customized
Primer Sets
PCR Amplification
with Microsynths
Primer Sets
PCR Amplification
with Custom
Primer Sets
PCR Purification and Equimolar Pooling
454 Sequencing
Bioinformatics Analysis
Microsynth AG | Schützenstrasse 15 | 9436 Balgach | Switzerland
E-mail: [email protected] | Web: www.microsynth.ch
Project Output:
User-friendly report including OTU
lists, taxonomy lists, rarefaction anaylsis and heat plots. Results can be used
in down-stream community analysis.
DNA Isolation
The customer needs to provide 50-300
mg sample material. Depending from
sample type and matrix, Microsynth will
apply the appropriate DNA isolation protocol. The DNA will be quantified by
spectrofluorimetry and checked for its
integrity on an agarose gel.
primer sets for the 16S rDNA region, Microsynth offers also the synthesis and
validation of customized fusion primer
sets. The template-specific primer sequences are given by the customer.
Based on our experience it is essential to
validate fusion primer sets because the
kinetics of long oligonucleotides in PCR
reactions can significantly deviate from
standard PCR reactions and may lead to
poor amplification of certain samples.
Microsynth will design the fusion primer
sets with different MIDs (according to the
number of samples) and test them for
successful PCR. Once developed, fusion
primer sets can serve the customer for
many other studies. A certificate, including primer sequences, chemicals, PCR
conditions and an agarose gel photograph, will be provided with each fusion
primer set.
for the first cycles. The resulting PCR
products serve as template DNA for the
second PCR step with fewer cycles. This
two-step PCR is made in order to increase
reproducibility and to improve the production of high-quality multiplex amplicon libraries (Berry & al., Appl. Environ.
Microbiol. doi: 10.1128/AEM.05220-11,
Barcoded primers used in multiplex amplicon pyrosequencing bias amplification). PCR products are purified, quantified with fluorescence spectroscopy
using Picogreen and pooled in equimolar
amounts.
ical repartition on the picotiter plate, as
well as the number of reads per sample,
is optimized for each project.
For metagenomic amplicon projects, and
if not otherwise specified by the customer, we perform an unidirectional sequencing (Lib-L adaptors).
nomic classification are calculated.
Detailed explanations about our
metagenomics data analyses pipeline
are given in the brochure: “Summary Bio-
informatics 16S rDNA Analysis” at the
NGS - Application part of our website.
Selection of Fusion Primers
Option 1: Microsynth Fusion Primers
Microsynth has developed and validated
some fusion primer sets for 16S rDNA.
The specificity of each fusion primer set
for the detection of different types of organisms is variable. The choice of adequate fusion primer sets will be discussed with the customer for each
project.
Option 2: Customized Fusion Primers
Besides our validated standard fusion
PCR Amplification
PCR amplification from 16S rDNA will be
made using either our fusion primer sets
or the customized fusion primer sets. The
PCR amplification is set up as a two-step
PCR and is performed with a state-ofthe-art high-fidelity polymerase. Template-specific short primers will be used
454 Sequencing
The sequencing is performed on our GS
Roche Sequencer (454 technology), using the Titanium FLX reagents. The
amount of samples pooled and the phys-
Bioinformatic Analysis
This analysis includes the sorting of the
reads according to the MIDs, quality trimming and mapping against known databases. Diversity measurements and taxo-
Need More Information?
Call us at +41 71 722 83 33…or
E-mail us at [email protected]
Microsynth AG | Schützenstrasse 15 | 9436 Balgach | Switzerland
E-mail: [email protected] | Web: www.microsynth.ch