Microbial Amplicon Sequencing

Description of Service

The Microbial Amplicon service offers full-length sequencing and taxonomic classification of amplicons generated from the 16S, 18S, and/or ITS marker genes. You can select the amount of data you need (3,000 raw reads for Standard service, 6,000 raw reads for Big, and 12,000 raw reads for Huge), and we recommend more reads for higher expected amplicon complexity and/or when higher statistical power is needed for analyzing experimental conditions (such as differential abundance analysis).

We sequence each amplicon with a full-length read using the newest long-read sequencing technology from Oxford Nanopore Technologies (ONT). We then align the raw sequencing reads to a comprehensive database of bacterial, archaeal, and eukaryotic species to determine their taxonomic classifications based on the 16S, 18S, and ITS sequences detected (see FAQ below, How is taxonomic analysis performed for the Microbial Amplicon service?). Taxonomic data is then compiled across all raw reads to generate metrics on the relative abundance of all species that comprise at least 0.1% of the microbial community, along with figures displaying the top 20 genera and species.

Data & File Types Delivered

When results are ready, you’ll receive an email to log in and download them from your Dashboard .

Raw read sequences (.fastq.gz file): Provides the sequences of all raw reads produced by your sample (3,000 raw reads for Standard service, 6,000 raw reads for Big, and 12,000 raw reads for Huge).
Taxonomy table (.csv file): Lists all taxa found in your sample that are above a relative threshold of 0.1% abundance. The list includes columns with the NCBI taxonomy ID, relative abundance, and full taxonomic ranks for each taxa from kingdom to species.
Stats (.csv file): Lists statistics about the sequencing reads (total number reads, total number bases, read quality, etc.)
Taxonomy plot by genus (.png file): Shows the relative abundance of the top 20 genera in your sample.
Taxonomy plot by species (.png file): Shows the relative abundance of the top 20 species in your sample.
Read length histogram (.png file): Displays the read lengths of all raw reads produced by the sample, including coloration to indicate the kingdom that the reads originate from (bacteria, archaea, protists, fungi, etc.)

Preparing Your Microbial Amplicon Samples

Submit your final purified, linear, double-stranded DNA amplicons in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water; avoid buffers containing DMSO whenever possible. Unpurified samples are likely to fail.

Microbial Amplicon samples are sequenced without primers, so please do not ship any primers with your samples or mixed into your samples.

STEP 1: Amplify & Purify Your Target Gene(s)

There are numerous approaches to generating 16S, 18S, and ITS amplicons, thus we do not provide specific recommendations. Please refer to published literature for protocols on amplification and purification, and see FAQ section below for further guidance.

STEP 2: Verify Concentration, Quality & Purity

DNA concentration of the purified amplicons should be measured with a fluorometric assay such as Qubit or equivalent fluorometric method (such as a plate reader) and normalized to 10-20 ng/ul . We recommend performing size verification on your full-length double-stranded DNA amplicons via gel electrophoresis. We recommend purity of 260/280 above 1.8 and 260/230 between 2.0-2.2. See FAQ section below for best practices to QC your amplicons.

FAQ

How do you sequence Microbial Amplicon samples?
We sequence each sample the newest long-read sequencing technology from Oxford Nanopore Technologies (ONT), including the following components:

Constructing long-read sequencing library using the newest v14 library prep chemistry , including end-ligation of your purified microbial amplicons. Your input amplicons are not fragmented or further amplified.
Sequencing the library with a primer-free protocol using the most accurate R10.4.1 flow cells (raw data is delivered in .fastq.gz format), producing a full-length sequencing read for each amplicon.
Aligning the raw sequencing reads to a comprehensive database of bacterial, archaeal, and eukaryotic species to determine their taxonomic classifications based on the 16S, 18S, and ITS sequences detected (see FAQ below, How is taxonomic analysis performed for the Microbial Amplicon service? ). Taxonomic data is compiled from all raw reads to generate metrics on the relative abundance of all species that comprise at least 0.1% of the microbial community, along with figures displaying the top 20 genera and species.

Microbial Amplicon samples are sequenced WITHOUT primers or amplification. Please do not ship any primers with your samples or mix primers into your samples.

How is taxonomic analysis performed for the Microbial Amplicon service?

We run the raw reads through a basic quality filter.
We classify each read against NCBI’s Targeted Loci database of bacterial, archaeal, and eukaryotic species using Kraken2 .
We estimate the relative abundance of each species using Bracken .
We use the above outputs to generate figures on read lengths and relative species abundance.

What data & file types will I receive for successful Microbial Amplicon sequencing?

Raw read sequences (.fastq.gz file): Provides the sequences of all raw reads produced by your sample (3,000 raw reads for Standard service, 6,000 raw reads for Big, and 12,000 raw reads for Huge). Please note that returning all raw reads means there is a small chance of demultiplexing error, so a few reads from your sample might be returned to another customer on the same sequencing run.
Taxonomy table (.csv file): Lists all taxa found in your sample that are above a relative threshold of 0.1% abundance. The list includes columns with the NCBI taxonomy ID, relative abundance, and full taxonomic ranks for each taxa from kingdom to species.
Stats (.csv file): Lists statistics about the sequencing reads (total number reads, total number bases, read quality, etc.)
Taxonomy plot by genus (.png file): Shows the relative abundance of the top 20 genera in your sample.
Taxonomy plot by species (.png file): Shows the relative abundance of the top 20 species in your sample.
Read length histogram (.png file): Displays the read lengths of all raw reads produced by the sample, including coloration to indicate the kingdom that the reads originate from (bacteria, archaea, protists, fungi, etc.)

How much sequencing coverage will I get for Microbial Amplicons ?
For Microbial Amplicon samples sent at the correct concentration of 10-20 ng/ul, we typically collect about 3,000 raw sequencing reads for Standard sequencing, 6,000 raw sequencing reads for Big sequencing, and 12,000 raw sequencing reads for Huge sequencing.

What is the limit of detection for the lowest abundance community members that can be detected with the Microbial Amplicon service?
We generate metrics on the relative abundance of all species that comprise at least 0.1% of the microbial community.

How accurate are the Microbial Amplicon sequencing results?
As per Oxford Nanopore's specs for the chemistry and flowcells we currently use for Microbial Amplicon sequencing, the raw read accuracy is typically >99.5%. We basecall the raw reads using ONT’s super-accurate basecalling model. We require a minimum raw read Qscore of 10 (90% accuracy) during sequencing.

What is your turnaround time for returning Microbial Amplicon sequencing results?
In the vast majority of cases, we deliver Microbial Amplicon sequencing results within 1 business day of receipt of your samples.

How do I get my results?
Once you sign in to your account, you can download your results from your Dashboard .

How do I amplify my target genes?
There are numerous approaches to generating 16S, 18S, and ITS amplicons, thus we do not provide specific recommendations. Please refer to published literature for protocols for amplification and purification.

Each sample you submit may contain one or more of these amplified marker genes (16S, 18S, and/or ITS) from a given biological specimen.
Sample barcoding is included in our service, so you do not need to use barcoded PCR primers.
Our service includes end repair, so note that any overhangs on your amplicons will be blunted (please avoid placing any critical nucleotides in the overhangs).

Please note that Microbial Amplicon service does NOT include extraction of genomic DNA from the biological source material, nor does it include PCR amplification of the target genes. We simply ligate ONT barcodes and sequencing adapters onto the purified 16S/18S/ITS amplicons that you ship to us, then sequence each molecule with a single full-length ONT read.

Can I submit non-full length amplicons for sequencing, e.g. amplicons generated from only the V4 variable region of the 16S gene?

Yes, we can sequence any microbial amplicons >400bp with this service, although please note that the accuracy of the taxonomic analysis will be lower for non-full length amplicons, so we strongly recommend amplifying the whole gene.

How do I quantify my amplicons?

Perform DNA quantification assay only with Qubit or equivalent fluorometric method, and submit your Microbial Amplicon samples normalized to DNA concentration of 10-20 ng/ul per sample.

If you opt to use a spectrophotometric method like Nanodrop , please be aware that these methods are much less accurate for measuring DNA concentration. Sending samples at too high OR too low concentration may adversely affect the library prep and/or sequencing reactions, possibly resulting in sequencing failure . Sending samples at the incorrect concentration (usually due to using Nanodrop for quantification) is by far the most common cause of sequencing failure. Accurate quantification and normalization are key!

How do I measure the quality and purity of my amplicons?

We recommend performing size verification on your full-length double-stranded DNA amplicons via gel electrophoresis. Purity may be assayed with Nanodrop or other spectrophotometric methods, and we recommend samples with 260/280 above 1.8 and 260/230 between 2.0-2.2.

For best results, samples should NOT contain any of the following:

RNA (RNase treatment is recommended during gDNA extraction)
Denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
Residual contaminants from the organism (heme, humic acid, polyphenols, etc.)

What guarantees do you provide?

Our ability to deliver the target outputs is directly dependent on the quantity, quality, and purity of the amplicons that are sent to us, so we do not guarantee results. If we are not able to achieve the minimum number of raw sequencing reads , our failure policy applies (see FAQ below).

Minimum raw read targets for successful Microbial Amplicon sequencing:

2,000 raw reads (Standard service)
4,000 raw reads (Big service)
8,000 raw reads (Huge service)

Why did my Microbial Amplicon sample fail?
For Microbial Amplicon samples, "failure" means that your sample did not produce the minimum number of raw sequencing reads (see FAQ above).

The outcomes of the taxonomic analyses will be highly variable depending on the specific microbial community, so these analyses are not used to determine the sequencing status (complete or fail) for this service. Status is strictly based on raw read yield.

Our low sequencing prices and fast turnaround times do not include any QC to verify that your shipped samples meet our requirements, or to determine why your samples failed to produce the target number of sequencing reads (or had low coverage). Although we do not provide definitive reasons for failure, by far the most common reasons are:

You submitted unpurified DNA.
Unpurified samples (with amplicons still in the original PCR reaction) are very likely to fail. Please purify your amplicons out of the PCR reaction using a spin column, AMpure XP beads , or equivalent method, and submit them in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water.
Samples contain persistent sequencing inhibitors or contaminants. Depending on the biological source material and extraction method, purified amplicons may still contain abundant or persistent impurities that require additional purification before library prep in order to obtain high quality sequencing results.
Samples are not prepared at the required DNA concentration of 10-20 ng/µl. The most common cause of this is using a Nanodrop to quantify DNA concentration. We strongly recommend using a Qubit or equivalent fluorometric method,

What is your policy when Microbial Amplicon samples fail?
It is relatively rare that we cannot achieve the read target for high quality purified amplicons, but some rate of failure is unavoidable. We do still charge for failed samples.

If you received less than:

2,000 raw reads (Standard service)
4,000 raw reads (Big service)
8,000 raw reads (Huge service),

then you are welcome to submit a rerun request through your Order Info page. We will evaluate whether your sample quality and quantity permits rerunning your sample.

Can you perform genomic DNA extraction for me, using my biological source material (soil, feces, swabs, etc.)?
Not yet, but this is currently under development!

Can you amplify the 16S gene for me?
Not yet, but this is currently under development!

Can you perform whole-genome sequencing and assembly of my metagenomic gDNA?
Not yet, but this is currently under development!

I have suggestions about the types of reports and figures you should generate from microbial amplicon results, or which databases you should use for processing the data. How can I let you know?
We appreciate your feedback on our services! Email us at support@plasmidsaurus.com to let us know your thoughts.