Microbiome

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.
  • For the Microbiome 16S Amplification & Sequencing service, this includes barcoded full-length amplification of the 16S gene with our in-house sequencing primers.
  • For the Microbiome Amplicon Sequencing service, this includes end-ligation of your 16S, 18S, and/or ITS amplicons. We do not further amplify your amplicons.
• 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.

As per Oxford Nanopore's specs for the chemistry and flowcells we currently use for Microbiome 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.

We run the raw reads through a basic quality filter (omit reads with Qscore <10, omit reads <400bp in length, and omit reads >3,000 bp in length), then classify each read against an rrnDB (v5.6) and NCBI Targeted Loci database of bacterial, archael, and eukaryotic species using emu (v3.5.1) to determine their taxonomic classifications. 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.

We generate metrics on the relative abundance of all species that comprise at least 0.1% of the microbial community.

If you would like us to attempt metagenomic DNA extraction from other types of materials that are not listed here, please contact us at support@plasmidsaurus.com to discuss options before order submission.

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).

For sample submitted at appropriate concentration, we collect:

• Standard = Up to 5,000 raw reads
• Big = Up to 10,000 raw reads
• Huge = Up to 20,000 raw reads
• Bronto (for 16S amplification and extraction services only) = Up to 500,000 raw reads

Nope! 16S Amplification & Sequencing samples with or without extraction are amplified with our own in-house primers for the full-length 16S gene, so please do not ship any primers with your samples or mixed into your samples.

There are numerous approaches to extracting metagenomic DNA from the source material (swabs, soil, feces, et cetera), so we do not provide specific recommendations. Any extraction method that yields high quality, high purity, high molecular weight, double-stranded gDNA that is free of nicks, gaps, breaks, and contaminants is suitable for this service. 

See sample prep for more information, or choose the extraction option and we can handle DNA extraction for you. 

Zymo DNA/RNA Shield™-preserved source materials for extraction cannot be shipped via your local US dropbox, but instead must be shipped directly to our Eugene lab. You can request a free shipping label during order submission or ship on your own to:

Plasmidsaurus
1850 Millrace Drive, Suite 200
Eugene, OR 97403

Due to customs regulations on the shipping of biological materials, this extraction service is currently only available to customers that are shipping samples within the US.

For Microbiome samples, "failure" means that your sample did not produce the minimum number of raw sequencing reads. 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:

• Samples contain sequencing inhibitors or contaminants
  • Depending on the biological source material and extraction method, extracted metagenomic DNA may still contain abundant or persistent impurities that require additional purification before library prep in order to obtain high quality sequencing results. Please purify your DNA using a spin column (we highly recommend Zymo OneStep PCR Inhibitor Removal Kit for metagenomic gDNA), with AMpure XP beads, or an equivalent method, and submit the purified gDNA in elution buffer (10 mM Tris, pH 8.5) or nuclease-free water
• Samples are not prepared at the required DNA concentration of 10 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

To achieve optimal sequencing results, please follow our recommended sample prep instructions. 

It is relatively rare that we cannot achieve the read target for high quality purified metagenomic DNA, but some rate of failure is unavoidable. We 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)
• 200,000 raw reads (Bronto 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.