Preparing the fratjuice for uBiome fratseq

uBiome — the 23andme of metagenomics / the microbiome — has graciously agreed to sponsor the 16S ribosomal RNA sequencing of our initial 5 fratjuice samples.

This issue will discuss our sample preparations for the uBiome kits.

Miranda Curtis and Elisabeth Bik from uBiome have been super helpful in providing sample advice and protocols. I've pasted the relevant portions of our email exchange below. Different messages are separated by horizontal rules.

From @dhimmel on 2017-07-21

As far as the sampling goes, I've done several of the uBiome sample sites in the past. However, I think our samples will be slightly different since they're liquid. Do you have a recommended protocol? Specifically, how many microliters should we pipette? Or should we use the cotton swabs? There is a small amount of sediment in the samples (presumably dirt). Should we avoid getting any of this sediment in the pipetted sample?

From Miranda on 2017-07-21

I also received some helpful insight from the lab regarding your questions:

It would be best if those samples would be stored in a freezer, not in a fridge. This will best preserve bacterial content. Some bacteria can still grow at low temperatures just above freezing, and the goal here is to prevent the original composition of the sample, not to have some bacteria grow and others die off.

In ideal circumstances, storing samples in a -80C freezer would be the best (most labs will have one), but if not available, a -20C freezer, such as used in a regular household would work as well.

Usually, when we collaborate on these sort of projects ahead of time our kits ship vials to the project leader containing a preservative fluid that can be used to mix some of this "juice" in. Once in this fluid, samples would be stable for several weeks at room temperature and can be shipped by regular mail.

Also for the recommended protocol we usually would go for swabs, as they are a good way to collect the most material from a surface area. Also, our collection procedure and tubes are designed for swabs.

In case of liquid, the answer would depend a bit on what the volume of the liquid actually is. Do you know how much volume was collected?

We can probably accommodate up to about 500 ul of liquid in our tubes, and vortexing the liquid to homogenize would work best.

However, if the collected amounts are much higher (>1 ml), it would be better to resort to a way to concentrate the sample. I would recommend to spin the samples down for ~2000 or 3000 g for 3 min, pipette off the supernatant (and save that), and resuspend the pellet in a bit of our stabilizing buffer and pipette back into the uBiome tube. This way, bacterial and debris pellets would be concentrated and the volume would be small enough to put into our tubes.

Alternatively, samples could be concentrated over 0.2 micron Nalgene filtering units, which is how in the past some lab teams have concentrated bacteria from 100 ml sea-water samples. The filter then can be put into the uBiome tubes.

In summary, the best method of choice would be dependent on 1) volumes of the collected samples and 2) equipment that the project has access to, such as swing out rotor centrifuges or vacuum pumps and filtration units.

But most of all, if the swabs and liquids have been stored in a fridge, moving these to a freezer ASAP would be of immediate importance for preserving these samples.

From @dhimmel on 2017-07-21

Depending on the sample, we have either 15 or 50 milliliters of juice in a conical tube. So we should have plenty of volume.

From @bemert on 2017-07-21

There's about 15mL - 50mL of each sample containing between 0.5mL - 2mL of sediment. Would that much sediment interfere with your extraction and if so, should we pass the sample through a more porous filter before pelleting? We really appreciate your help.

From Miranda on 2017-07-22

It shouldn't interfere with the extraction, but you can probably go for multiple approaches, where you should take different types of aliquots from each sample. But It is hard to tell up front what the best sample type will be; some approaches might be too concentrated or not concentrated enough. So in the end, not all approaches might work, but hopefully at least one of the three will work for all samples. In each kit, there will be 2 tubes in total in case you want to try an additional method.

Here is a proposal for 3 different aliquots per sample provided from our lab:

Swab, presumably wet with visible material on it -> transfer directly in a uBiome tube, swirl around vigorously for 1 min, remove swab (should probably save swab)
Tube with fluid: Shake the 15 or 50 ml tube with fluid to homogenize (or vortex for 5 sec). Immediately take 300 ul of fluid with particles, and pipet that into second uBiome tube. For this, a large orifice pipet tip might work well. It's probably fine if very large particles are avoided, but anything that will fit into a pipette tip should be transferred. Mix to homogenize with the uBiome stabilization buffer.
Before the fluid settles after taking the 2nd sample, take equal amounts, e.g. 5 ml of each sample, transfer each to fresh 15 ml tube, spin down 3000 g for 3 min. Pipette off supernatant (and save in another fresh tube for future use) but leave a tiny bit, about 300 ul in the tube. Use that to resuspend the pellet and transfer the whole 300 ul with sediment to another uBiome tube. Mix to homogenize with the uBiome stabilization buffer.

Once in uBiome tube, the samples will be stable at RT for weeks.

At around 5:30 pm on 2017-06-22, Ben Emert performed an initial preparation of the samples for when the uBiome kits arrive. I'll let Ben fill in the details. I primarily just watched in fascination of his wet lab rigor and adroitness.

We ended up following aliquot methods 2 and 3 as suggested by Miranda above. We didn't want to wait any longer before freezing the samples, as its already been ~11 days since sample collection. The samples were maintained at refrigerated temperature (approximately 4 degrees Celsius).

For method 2, we took 300 ul of mixed juice and froze it at -80 C. For method 3, we concentrated the solids using a centrifuge. Then we resuspended the pellet after removing most of the supernatant. We froze these sample at -80 C. So we now have unconcentrated (method 2) and concentrated samples (method 3).

We separated the remaining juice into a small amount for continued refridgeration, in case we want to plate it soon. The remainder, we're freezing in a common freezer for potential later use.

As Daniel mentioned, on 2017-06-22 we prepared two aliquots of each sample for shipment to uBiome. First, each sample was mixed by vortexing and inverting the tubes, then 300uL were transferred to individual 1.5mL eppendorfs. 5mL of each sample was then transferred to 15mL falcon tubes and centrifuged at 3,000 x g for 5 minutes at room temperature. We removed approximately 4.7mL of supernatant and resuspended the pellet in the remaining liquid volume before transferring to 1.5mL eppendorfs. All sample were then frozen at -80ºC.

Before moving the remaining sample to Danny's freezer, we removed 1mL of each sample for storage at 4ºC with the hope of plating these aliquots in the near future.

Last week on 2017-07-12, the five sample kits arrived from uBiome (UPS Tracking 1Z30222R0315196661). The order number was 2890147 dated 2017-06-30, which contained five One Time uBiome Explorer Gut Kits (Item SKU RL-X5TI-09NL, Barcode 210599).

Today at around 3:30 PM EDT on 2017-07-20, @bemert and I transferred the samples into the tubes. Each of the five kits contained two tubes: one labeled "Gut" and one labeled "Spare". @bemert transferred the concentrated fratjuice to the Gut tubes and the unconcentrated fratjuice to the Spare tubes. For both sample types, @bemert pipetted 300 microliters into the uBiome tube. Then I vortexed the tubes for ~30 seconds and shook them by hand as well. See kits.tsv for the details of the sample to uBiome kit/tube mapping.

Here's the tubes looked like when we had finished transferring the fratjuice:

2017-07-20_ubiome-tubes

Update: On 2017-07-21, I put the 10 tubes in the return mailer for kit 592-185-324. The USPS Tracking Number is for the mailer is 9201990166692701144771. Since Philly is quite hot this time of year, I was slightly worried about the samples sitting in a USPS collection box. On the samples' endurance to heat, Elies commented:

That should be OK. The uBiome tubes are filled with a stabilization buffer that will preserve the community as soon as the sample touches the buffer. But maybe put them in a dropbox that is not in the full sun?

So I chose a dropbox in the shade and dropped off the sample around 4:45 PM. Luckily, USPS collected the parcel later that day, and the parcel is now on its way to San Francisco via Priority Mail™.

dhimmel / fratjuice