PRE-SEQUENCING – COLLECTION
Do you prefer that samples are sent frozen?
We recommend samples remain frozen in the -80 until ready to be shipped, at which time they need to be shipped on dry ice. The only exception is when samples are being collected at home and can be done so using a manufactured collection device with a stabilizing buffer for room temperature stability.
Do you provide collection kits?
We do not provide collection kits however, we do recommend using the 1) Becton-Dickinson, BBL CultureSwab EZ II which includes a double-swab encased in a rigid non-breathable transport tube 2) collection devices from Norgen Biotek and 3) collection devices from DNA Genotek.
How much (raw frozen) sample do I need to send?
- Fecal swabs: Ensure the swab is discolored
- Skin swabs: swab(s) rubbed back and forth vigorously 50 times (for 30 seconds)
- Oral cavity swab: swab(s) rubbed behind molar teeth, roof of mouth and cheek vigorously for 3 minutes.
- Rodent fecal samples: 2-3 frozen pellets
- Tissue: 1.00 g or approximately 0.4mL of tissue
- Soil sample: 1.00g of soil in a cryo-vial or microfuge tube
For all other samples please ask your account manager for guidance.
For samples provided in a manufactured collection device please follow manufacturer’s instructions on sample quantity.
Do you accept low biomass samples?
Depending on the sample type we require larger sample mass to be sent in order to account for troubleshooting steps. While we cannot guarantee that we will be able to amplify the amplicons of interest from your samples, we will do our best to work with these samples and troubleshoot where necessary.
Do I need a second sample to run Short Chain Fatty Acid analysis?
Yes, please provide a separate tube with a minimum of 150mg of stool for SCFA analysis.
Do you accept extracted samples?
We prefer to do extractions inhouse but if you are sending extracted samples we require concentrations and volumes for each sample.
How much extract do I need to send?
The absolute minimum we need is 100ng total. Ideally, we want 10ng/ul of DNA that has been quantified using Qubit or Picogreen (or similar), but not nanodrop.
PRE-SEQUENCING – STORAGE AND TRANSPORT
Can an at-home donor store their sample in the freezer?
While some sample types are easier to collect at home, using a swab or collector, the at-home freezers have inherent freeze-thaw cycles that are not ideal for stabilizing the microbiome. It is highly recommended that samples be transported (on dry ice) to a -80 as quickly as possible.
If your donor is using a collection kit with buffer for room temperature stabilization the sample need not go in the freezer, but it recommended the donor send the sample to the clinic or laboratory so not to forget or lose.
How long can a sample be stored at -80?
Samples may be stored in the -80 indefinitely, although it is preferable that samples be homogenized, extracted and aliquoted for long term storage.
If your sample is stored at room temperature in a buffer, be sure to follow the manufacturer’s recommendations for room temperature stability, and place the samples in the -80 within the manufacturer’s guidelines.
Do you have storage capacity at your lab?
We have limited space and request that you batch and ship in quantities no smaller than 25 samples at a time.
How long do you keep my samples? Can you send them back to me?
We keep the samples on hand for 3 months at which point we discard them. This is a good option ONLY if you are considering future assessment using the same samples. Otherwise, we prefer to send the samples back to you.
Should we plate our samples?
Plating is not necessary unless you have a full 96 samples (or multiples thereof) to be analyzed. In such a case, plating is only helpful if you are sending extracted fecal samples, or are plating cut swab heads in a deep well plate. Plates need to be sealed, each well ID’ed and logged in the sample manifest and plate template (provided by your account manager), and shipped on dry ice.
How should we send samples if not plated?
For any of the samples you are sending us, we prefer that you ship on dry ice. Shipping instructions can be found on our website. Please ensure that samples are packed with enough dry ice to last the duration of the shipment, and that samples are sent on Mon-Wed to avoid any delays at customs over the weekend.
What do I need to send along with my samples?
- All samples must be labeled according to the identification numbers provided on the sample manifest. Please ask your representative for a unique sample manifest for submission of your samples.
- Microbiome Insights requires the sample manifest to be completed and returned electronically prior to receiving your samples.
- Please package your samples in the same order as listed in the sample manifest. If Microbiome Insights is required to sort and reorder samples prior to extracting, additional fees may result.
Where should I send my samples?
Please send samples to:
Attn Ben Tantika
13520 Crestwood Place, Unit 12
Richmond, BC, Canada, V6V 2G3
If shipping soil, samples please let your Microbiome Insights representative know.
PRE-SEQUENCING – EXTRACTION
What extraction chemistry do you use?
The extraction process for Amplicon Sequencing, qPCR and Shotgun Metagenomics are conducted in the same fashion using the MO BIO Powersoil DNA extraction kit optimized for both manual extractions and automated extractions on the ThermoFisher KingFisher robot.
Extractions for both SCFA and for Calprotectin testing are unique from the above and from one another.
Do you use bead beating in your extraction process?
The MO BIO Powersoil DNA protocol is followed, with the addition of a bead beating step to facilitate lysis of particularly robust microorganisms.
What is your QC process for sequencing?
DNA quantification and quality checks are done via Qubit. We use the high-fidelity Phusion polymerase for amplification of 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. Samples with failed PCRs (or spurious bands) are re-amplified by optimizing PCR conditions.
How do you run your PCR?
18S V4 (Eukarya) or ITS2 (Fungi) 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. 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.
Do I need to pay per plate?
We run 4 96-well plates per sequencing run in the Illumina Miseq. As such we multiplex our samples allowing us to charge ‘per sample’ rather than ‘per plate’. Samples with low biomass will be run on a plate with fewer samples to attribute more reads to these samples. As such we charge total number of wells for your use.
How do you avoid batch effects?
One way to successfully reduce human error and avoid batch effects is to wait until all samples in the study are complete, and run them at the same time. If samples are collected over an extended period samples can be run per timepoint if necessary.
SEQUENCING – (ITAG) AMPLICON
Which amplicons do you run?
We can address taxonomic profiling for prokaryotic communities (16S V4, V1-V3 or V4_skin), eukaryotic communities (18S), fungal communities (ITS2), and archaeal communities (16S V4-V5) on the Illumina Miseq using the version 3 chemistry.
Why did you select the 16 V4 region?
We selected the 16S V4 region for prokaryotic sequencing as there is optimal overlap of bp length (250bp) from this region as is relates to the version 2 Illumina Miseq chemistry. As such, there are fewer errors using this workflow.
Why do some labs select other primers?
V1-V3 may produce better classification for skin microbiota; conversely the V4 region is less suitable for amplifying skin taxa and classification. The 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?
The average number of reads per sample is close to 15-20K (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 also 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 also not toss out samples with low reads as there are likely biological factors at play. Course-grained comparative analysis (ex Beta diversity) are relatively robust to low reads.
What is qPCR?
Quantitative PCR (qPCR, also known as Real-time PCR) is a method that measures the number of copies of a DNA region defined by PCR primers. This method is a great complement to microbiome sequencing, which targets the whole community, as it provides the absolute abundance of specific populations of interest.
When is it optimal to run qPCR?
If you are interested in identifying ‘how much’ bacteria is in the sample (rather than breaking it down by taxonomy) we can run a qPCR which gives us a single number of total bacterial quantification. This is also a good way to assess whether there is enough bacteria to amplify in your sample before running on 16S, particularly in the case of low abundance samples.
Can I look at all bacterial species?
Yes but this will give you a total quantity of ALL the bacteria in the sample. If you are interested in the relative abundance of each bacteria we suggest 16S amplicon sequencing.
SHOTGUN METAGENOMIC SEQUENCING
When is it beneficial to use Shotgun Metagenomic Sequencing?
While amplicon sequencing provides a good basis for relative abundance of the genus/phyla level per amplicon as well as alpha and beta diversity, your research may require investigation at the species level and/or at the functional level. If so, this is a good option for your study.
Will I only see bacteria?
Using Shotgun Metagenomics you will see anything that can be amplified in this sample. This refers to all DNA that can be amplified in the sample, including non-microbes.
Can I use Shotgun Metagenomics for viral analysis?
This test is not optimized for viriome work, but we can assess the nature of your study to see what would be applicable.
What do you use for Shotgun Metagenomic Sequencing?
We have access to both the Illumina Nextseq and Illumina Hiseq for Shotgun Metagenomic Sequencing. In particular, we can run your samples on the 1) Nextseq Medium, 2) Nextseq High, 3) HiSeq rapid run 2×100 or the 4) HiSeq rapid run 2×250
|NextSeq Medium||MAX 31||HiSeq rapid run 2×100||MAX 48|
|Total reads per run (M)||260||Total reads per run (M)||600|
|NextSeq High||MAX 96||HiSeq rapid run 2×250||MAX 120|
|Total reads per run (M)||800||Total reads per run (M)||600|
What is the best option for me?
In choosing the right platform for you, we would need to assess the sample type, number of samples, and desired number of reads per sample (coverage). With this we can determine how to achieve the best combination for your samples.
Each platform has a maximum number of samples per lane. We can either determine pricing by the total cost per lane divided by the number of desired samples OR we can determine the optimal number of samples to place in each lane based on the total number of reads per lane divided by the desired coverage per sample.
How do you prepare your libraries?
Whole DNA is prepared for sequencing using the Illumina Nextera XT DNA Library prep kit. Samples are barcoded and mixed together for sequencing. The mix is then sequenced using Illumina technologies for either high-output or medium-output.
How are sequences processed after sequencing?
Paired reads are concatenated and then processed to remove adaptors, barcodes, low-quality reads as well as contaminant sequences (typically host DNA). Sequences are then compared to phylogenetic and functional databases to obtain taxonomic and functional profiles.
SHORT CHAIN FATTY ACID TESTING
What is Short Chain Fatty Acid testing and when is it used?
Short Chain Fatty Acids (SCFA) are the products of fermentation of insoluble fibre from diet (eg. cellulose, resistant starch) by the bacteria in the gut. These fatty acids have been shown to play an important role in regulating metabolism in the gut and are closely associated with gastrointestinal diseases such as irritable bowel syndrome and other conditions such as obesity. By quantifying SCFA in stool, one can monitor gut health and inflammation.
What method do you use to test for Short Chain Fatty Acids?
Short Chain Fatty Acids are extracted from stool in an aqueous solution, analyzed in a gas chromatograph (GC) coupled with a flame ionization detector (FID), using a Thermo TG-WAXMS A GC column. The analytes are quantified against a series of stock standard solutions.
Which Short Chain Fatty Acids do you report on?
We report data for acetic, propionic, butyric, valeric, isovaleric, hexanoic, isobutyric and heptanoic acids.
Can I bundle Amplicon Sequencing with Short Chain Fatty Acid Analysis?
Yes you certainly can, if there is enough sample to obtain two unique extractions.
What sample type would you analyse for this type of test?
We can use human fecal samples (frozen or in the DNA Genotek OMNIgut collection kit), and rodent fecal pellets (frozen). We have not run this sample on any other sample type, nor would it be necessarily relevant since SCFAs are particular to the gut.
What do you report for Short Chain Fatty Acid Analysis?
The output of the GC is a clearly depicted chromatograph with concentration peaks for each short chain fatty acid.
What do you report for iTag Amplicon Sequencing?
For each of the amplicons that we target, our report includes relative abundance per sample, ordination and beta diversity.
How do you assess the relative abundance?
Raw Fastq files are quality-filtered and clustered into 97% similarity operational taxonomic units (OTUs) using the Mothur software, to result in taxonomic assignment. We then use the Greengenes database to link the amplicon sequences to hierarchical taxonomic schemes.
At what level are we able to detect the relative abundance?
Using the Illumina Miseq version 2 chemistry with the amplicons on our test panel, we can detect the relative abundance at the family, genus and phyla level, with some species level identification.
What do you report for the Shotgun Metagenomic Sequencing?
In a general sense, sequences of all amplified DNA – both microbial and non-microbial – are binned. Protein coding and community functional diversity profile are identified. A report is provided based on the scientific questions, with a full dataset available for manipulation.
What do you report for qPCR?
For this type of test we are reporting on the total bacterial content (quantification) and/or the quantification of species (primers) amplified per sample.
Am I able to forgo the bioinformatics and just have you send the FastQ files?
Yes, we can do this. However, our bioinformaticians are experts in this field and have the capabilities to provide you with a fully comprehensive, publication quality report for a margin of the cost. They assess the data from the bioinformatic, statistical, and theoretical angle to make sense of your results.