Microbiome Technical Documentation
Technical details
Plasmidsaurus Microbiome Sequencing allows you to get a snapshot of your microbial community composition.
Sequencing technology
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.
Bioinformatics analysis
We use the latest Super-Accurate basecalling model and run the raw reads through a basic quality filter to omit reads with Qscore <10, omit reads <400bp in length, and omit reads >3,000 bp in length. We 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.
Service levels and coverage
| Sample inputs | ||||
|---|---|---|---|---|
| Service Level | Read Depth/Sample | Microbiome 16S Amplification & Sequencing with DNA Extraction | Microbiome 16S Amplification & Sequencing | Microbiome Amplicon Sequencing. Cleanup option available. |
| Standard | Up to 5,000 | Raw environmental samples. See sample prep for more info. | 10 µL of extracted gDNA at 10 ng/µL | 10 µL of amplified DNA at 10-20 ng/µL |
| Big | Up to 10,000 | 20 µL of extracted gDNA at 10 ng/µL | 20 µL of amplified DNA at 10-20 ng/µL | |
| Huge | Up to 20,000 | 40 µL of extracted gDNA at 10 ng/µL | 40 µL of amplified DNA at 10-20 ng/µL | |
| Bronto | Up to 500,000 | 40 µL of extracted gDNA at 10 ng/µL | Not available | |
Data deliverables and file types
- Raw read sequences (.fastq.gz file): Provides the sequences of all raw reads produced by your sample (up to 5,000 raw reads for Standard service, 10,000 raw reads for Big, 20,000 raw reads for Huge, and 500,000 raw reads for Bronto). 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.
- Read taxonomic assignment (.tsv file): Lists all reads and the taxa they were assigned to.
- 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.)
Troubleshooting
Likely causes of microbiome sequencing failure
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.
Guarantees and rerun policy
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.