A Method for Targeted 16S Sequencing of Human Milk Samples

The overall goal of this method is to amplify 16 SRRNA from human milk and other low biomass samples using a semi-automated protocol. This method can help answer key questions in the development and perturbations of the human microbiome as it relates both to health and disease. The main advantage of the technique is that it's semi-automated, relatively easy to perform, and adaptable to multiple sample types including secretion, swabs, and stool.

After thawing and alloquating whole milk according to the text protocol, spin the samples at 5000 times G and four degrees Celsius for 10 minutes to pellet the cells. Use a plastic spatula or large bore pipette tip to remove the top fat layer. Then without disturbing the pellet, remove all the supernatant, except for 100 microliters.

With one milliliter of PBS, re-suspend and wash the pellet. Then prepare a negative control by adding one milliliter of sterile PBS to a five milliliter tube. Transfer the suspension to a clean one point five milliliter centrifuge tube and spin it at 5000 times G at room temperature for one minute.

Then use a 1000 microliter sterile filtered pipette tip to discard the entire supernatant fatty layer. To disrupt and homogenize the sample, add 600 microliters of lysis buffer containing beta mercaptoethanol to the pellet and transfer the suspension to a bead tube. Prepare one positive control with lysis buffer and 20 microliters of the bacterial mock sample.

Vortex all the bead tubes vigorously for 15 seconds. Then heat the samples on a temperature controlled vortexer at 37 degrees Celsius and 700 to 800 RPM for 10 minutes. Load the tubes into the automated sample disrupter adaptor set.

Then bead beat the samples at 30 hertz for one minute. Disassemble the adaptor set and manually switch it with the sample plates from the left robotic arm to the right robotic arm of the instrument. Bead beat the tubes at 30 hertz for another minute then centrifuge the tubes at 17, 200 times G and 25 degrees Celsius for three minutes.

Using a 200 microliter pipette, remove all of the supernatant, taking care not to take any of the fatty layer at the top or the bead residue at the bottom of the sample. Apply the supernatant to homogenizer columns. Then centrifuge the columns at maximum speed and 25 degrees Celsius for three minutes.

Transfer 350 microliters of eluid to a two milliliter manufacturers microcentrifuge tube. For isolating DNA according to the text protocol, cut off the lids and smooth the edges of the individual spin columns for DNA and RNA. Insert the DNA spin column without the collection tube into the rotor adapter.

Then discard the collection tube. Next, insert the vortexed two milliliter tubes into a shaker, following the automated DNA/RNA purification instrument's loading chart per the manufacturer's instructions. Label one point five milliliter collection tubes and insert them into rotor adapters.

Then set the rotor adapters into the centrifuge following the automated DNA/RNA purification instrument's loading chart. After inserting filter tips, RNase free water, and the reagents into the instrument's reagent block, close the instrument's lid and run the custom DNA purification program on the instrument. When the reaction is complete, collect the sample containing tubes from the shaker, and if any sample is left, store it at minus 20 degrees Celsius for short term storage or minus 80 degrees Celsius for long term storage.

After preparing the work area, and the PCR master mix, according to the text protocol, load 50 to 100 microliters of DNA samples into a 32-well instrument sample adapter, following the 96-well plate map. For each PCR plate, set up two negative controls by pipetting 30 microliters of PCR water in a clean one point five milliliter collection tube. Then place all the samples on the 32-well instrument sample adapter with caps locked in the open position.

On the instrument's reagent adapter, remove the cap of each reverse primer with specific bar code numbers one at at time. To load the robotic liquid handler, place the reagent adapter in position B1.In order to avoid an edge effect, carefully place one edge of the adapter against the grip side and slowly bring the other edge down. Make sure to push on all the corners of the adapters.

Place the sample adapter in position C1, making sure to push on all the corners of the adapters. Then vortex the five milliliter master mix. Open the cap and place it in position A on the instrument's master mix and reagent block.

Place the PCR plate on the 96-well instrument's adapter that is intended to hold half skirted PCR plates. Then start the run and save it as a new file. When the run is complete, verify that there were no errors.

Then use 12 or eight strip caps to seal the plate. Vortex vigorously and use a 96-well plate spinner to spin down the plates for one minute. Finally, place the plate on ice until it is moved to the post amplification room and loaded onto the thermal cycler.

Following PCR amplification, carry out quality control and library preparation according to the text protocol. The protocol's first QC step follows PCR amplification of the 16SV4 region. As shown here, one microliter of PCR product from each sample, was analyzed by electrophoresis to confirm that it was within the expected size range of 350 to 450 base pairs.

Some human milk samples generated lower amounts of specific product, suggesting either low levels of extracted microbial DNA in those samples or carryover of PCR inhibitors during extraction. Quantitation of specific product for each sample was essential for determining its required volume for equal molar pooling of samples for sequencing. A pooled library for targeted sequencing is usually dominated by a specific PCR product.

If there is a significant amount of nonspecific product in the library, a gel purification step should be added to the workflow. Sequencing results demonstrate high diversity in the taxa associated with the human milk microbiome and variability in the number of sequencing reed counts for each sample. In contrast, the taxa composition and reed counts for the bacterial mock, were comparable to results obtained for the mock in previous workflow runs, suggesting that the observed variability for the human milk samples is an authentic experimental result and not a function of intrinsic workflow variability.

If leaving a sequencer running overnight this technique can be done in two days, for 12 to 24 samples, if all samples amplify into the initial PCR run. If not, another shell day is needed. When attempting this procedure, it is important to travel only from the pre-PCR area to the post-PCR area to avoid contamination.

It is also critical to change gloves frequently to avoid any cross contamination. Don't forget that working with human samples can be hazardous and precautions, such as proper protective equipment and bio safety cabinets, should always be used.