Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The overall goal of this procedure is to collect and process complex host associated samples such as cystic fibrosis sputum, to study the viral and microbial communities using metagenomic and meta transcriptomic approaches. This is accomplished by first having the subject inhale, 7%hypertonic saline via a nebulizer, and then collecting induced sputum samples over a 30 minute time period. Next, the sample is immediately homogenized and equally distributed into four tubes labeled as viral metagenome, microbial metagenome, meta transcriptome, and extra sputum.

Next, the meta transcriptome tube is sealed, placed horizontally and the sputum homogenized at medium speed for 10 minutes. Finally, individual aliquots are processed separately to isolate and purify viral or bacterial cells prior to nucleic acid isolation for metagenomic studies. Ultimately, the nucleic acids isolated are used for high throughput sequencing library preparation to generate metagenomics and meta transcriptomics libraries.

The main advantage of this procedure is that it provides a comprehensive workflow to process complex host associated sample for the study of viral and bacterial communities at the DNA and RNA level. Visual demonstration of this procedure is critical as the sample pretreatment steps are important to ensure proper handling of the sample while the viral and microbial cell purification steps are difficult to learn Due to the complexity in procedure Prior to sample collection, label four 15 milliliter tubes per sample with the following, the first with viral metagenome, the second as microbial metagenome number three with meta transcriptome and tube number four as extra sputum. Add two milliliters of 0.1 millimeters silica beads into the tube labeled meta transcriptome, followed by six milliliters of guine isothiocyanate based RNA lysis buffer, or GITC lysis buffer.

Following the sample collection procedure outlined in the text protocol and using pre wade sample cups, estimate the sample volume by weighing the cup after sample collection and subtract the weight of the empty cup. For the remaining weight, one gram will be equivalent to one milliliter of sample. If the volume of the sample is less than eight milliliters, add the appropriate volume of 0.02 micrometer filtered one XPBS to bring the final volume up to eight milliliters.

Then immediately use a three milliliter syringe to homogenize the sample until no visible clumps remain within the sputum using the same syringe immediately draw up two milliliters of sputum and inject it into the micro transcriptome tube containing silica beads and GITC lysis buffer. Close the lid and use para film to securely seal the lid To avoid leakage, use a vortex, sir to immediately homogenize the sputum at medium speed for 10 minutes. Using the same syringe aliquot, two milliliters of sputum each into the tubes labeled vira metagenome and microbial metagenome, and transfer the remaining sputum from the sputum cup into the tube labeled extra sputum.

Store all tubes at four degrees Celsius or on ice and transport to the lab if necessary to generate a viral metagenome. Following the preparation of buffers and solutions. Pretreat the samples by adding 0.02 micrometer filtered saline magnesium buffer to a total volume of six milliliters.

To aid in mucus dissolution, add six milliliters of 6.5 millimolar of DTT to each sample. Vortex vigorously to mix and incubate for one hour at 37 degrees Celsius. After vigorously vortexing the samples again spin at 3056 Gs and 10 degrees Celsius for 15 to 20 minutes.

Collect each supernatant into a new 15 milliliter tube. Next, use a point 45 micrometer filter mounted on a syringe to filter the supernatants into new 15 milliliter tubes. Remove 100 microliters of sample into a micro fuge tube and then perform chloroform treatment.

Extract the supernatant and perform DN one treatment. Add an equal volume of 4%para of formaldehyde to fix the sample. For epi fluorescence microscopy, refer to the text protocol for a catch-all viral particle enrichment approach.

Otherwise, using freshly prepared caesium chloride solutions, set up a caesium chloride gradient by loading one milliliter of 1.7 grams per milliliter of caesium chloride solution into each tube, followed by one milliliter of 1.5 grams per milliliter, one milliliter of one point 35 grams per milliliter, and finally, one milliliter of 1.2 grams per milliliter of cesium chloride solution. Finally, load six to eight milliliters of sample into individual gradient tubes. Mark individual layers to denote the location of each fraction.

Then balance each opposing pair of twos to within one milligram. Carefully lock each tube into the spin bucket. Load onto the rotor and centrifuge at 82, 844 Gs and four degrees Celsius for two hours.

Following centrifugation, carefully remove the tubes from the holder without disrupting the density gradients with an 18 gauge needle attached to a three milliliter syringe. Ensure the open end of the needle face up. Pierce the tube just below the 1.5 gram per milliliter density layer and slowly draw approximately 1.5 milliliter of solution into the syringe.

Slowly remove the needle and allow the remaining fraction in the tube to drip into a new 15 milliliter tube through the puncture. Label this as upper fraction waist Transfer the solution in the syringe containing the V ps into two new micro fuge tubes. Once buffers and solutions have been prepared, according to the text protocol, add five volumes of point 22 micrometer filtered one XPBS To dilute each homogenate under a chemical hood, add beta MAR capta ethanol to a 2%volume to volume final concentration, and rock the mixture at room temperature for two hours following the incubation.

Spin the sample at 3056 GS and 10 degrees Celsius for 15 minutes. After discarding the supernatant, use 10 milliliters of molecular grade water to resuspend the pellet and incubate at room temperature for 15 minutes before repeating the wash. After a final spin, and once the SUP natin has been discarded, use five milliliters of one XDNA buffer to resuspend the pellet before adding 15 microliters of ds.

One per milliliter of sample Incubate at 37 degrees Celsius with repeated mixing for two hours. After spinning the samples and discarding the snat, use 10 milliliters of SE buffer to resuspend the pellet. Then spin the sample and repeat the wash.

Use two milliliters of SE buffer to resuspend the pellet and transfer the suspension into two micro fuge tubes. Then spin the tubes at 16, 000 GS at room temperature for 15 minutes. Finally, after removing the supernatant, use a genomic DNA extraction kit following the instructions for gram-positive bacteria.

To isolate the DNA previous results to generate viral metagenomes are summarized here. The viromes contain little human derived sequences with only one sample containing 70%human DNA sample CCF four A was omitted from the density gradient ultracentrifugation step, and as a result, it contained greater than 97%human derived sequences. This figure shows a fluorescent image of a typical CF sputum sample before and after density gradient ultracentrifugation.

Clear vlp were observed in the absence of large particles following the density gradient separation seen here. Seven daily sputum samples were collected from a single cystic fibrosis patient who began oral antibiotics. After day three of collection, the total yield of hypotonic lysis derived or HL DNA collected from each sample ranged from 210 nanograms to greater than five micrograms from Illumina sequencing Libraries generated for each sample.

All but one library yielded more than 1 million sequences and more than 85%high quality sequences were retained upon data pre-processing. Using print seq software, the total amount of human derived sequences ranged from 14%to 46%As shown here, the microbial profiles of the subsamples are highly similar to the samples after two hypotonic lysis treatments. In addition, the second hypotonic lysis increases the fraction of non-human sequences by six to 17%within the metagenomes.

Don’t forget that working with clinical samples and potential pathogens can be extremely hazardous. Personal protective equipment should always be used at all time during sample handling. Good molecular biology and microbiology techniques are required while performing this procedure to ensure no contamination.

Following this procedure, you should be able to perform viral and microbial metagenomic, as well as meta transcriptomic studies from complex samples such as CF sputum.