The overall goal of this procedure is to isolate single varians from a mixed consortia and to sequence the obtained genomic material to identify viruses from a variety of environments without the need for cultivation. This is accomplished by first isolating the single varians using flow's cytometric sorting onto aro beads. Single ion capture is then validated by confocal microscopy.
Next, the genomic material is isolated from the ion's and amplified using multiple displacement amplification or MDA to generate the quantity of DNA needed for sequencing. Finally, the viral DNA is sequenced using high throughput technologies to generate excess coverage of the virion genome substantially aiding in the genome assembly and annotation. Ultimately, bioinformatic techniques aimed at using sequence sub sampling approaches to increase con size allow for analysis of isolated vons.
This method can help answer key questions in the field of viral ecology by giving genomic context to the vast pool of annotated OC plankton genes, as well as enable the discovery of new genomes from varied ecosystems. Generally, individuals that are new to this method may struggle because amplification and isolation of single ions is pushing the limits of current technology. We first had the idea for this method when we were assessing the use of MDA on Vero plankton communities and realized it was possible to sort a single viral particle and sequence and therefore assemble a full viral genome from these complex communities To sort viruses using a flow cytometer equipped with custom forward scatter photomultiplier tubes or F-S-C-P-M-T begin by diluting the viral particles in 0.1 micron filtered tris, EDTA for a mixed assemblage containing T four and lambda phage particles set the F-S-C-P-M-T to 1000 and the SSC to 200 to maximize the signal to noise ratio.
After running control samples, run about 100 microliters of the sustained viral suspension to a total of 5, 000 events. Then use the cyber green one and F-S-C-P-M-T plots to place a tight gate in the center of the viral particles. Now add five microliters of 1%low melting point aros in TBE buffer cooled to 37 degrees Celsius to each well on A-P-T-F-E microscope slide and then load the slide into the flow cytometer to capture the sorted viral particles.
Begin sorting, capturing 10 or more viral particle events in some of the wells to aid in the detection of the depth of viruses during confocal laser scanning microscopy. When sorting is complete, remove the slide from the flow cytometer and add five more microliters of low melting point aros cooled to 37 degrees Celsius to each well. Embedding the viral particles for single varion.
Set the confocal laser scanning microscope to 488 nanometers. Excitation then image the embedded particles using a 63 x long working distance objective to verify that only a single particle is present and that multiple viruses are not stacked on top of each other. Then once the wells with single viruses have been identified, use a razor blade to remove the desired agros beads from the slide and place them in a sterile PCR tube.
Place the tube in the heat block of a thermocycler set to 94 degrees Celsius for three minutes to lice the viral particle in situ. Then after performing a modified MDA transfer the genomic DNA to 1.7 milliliter einor tubes and add a one 10th volume of three molar sodium acetate in TBE buffer To purify the amplified genomic material, place the samples at 55 degrees Celsius for 10 minutes to dissolve the aros and then move the tubes to 42 degrees Celsius to reduce the temperature prior to adding the enzyme. Now add two units of beta aase to each tube and incubate the tubes for two hours.
Then add one volume of a buffer saturated phenol solution to each tube. Mix vigorously and centrifuge the tubes for 15 minutes at 3, 500 times G.At room temperature, transfer the DNA containing SUPERNATANTS to a new 1.7 milliliter einor tube. Next, add one volume of 100%isopropanol and one microliter of glyco blue to the tubes and invert the tubes to mix after centrifuging the tubes again.
This time for 60 minutes at 28, 000 times G and four degrees Celsius. Decant the isopropanol, making sure not to disturb the pellet and add 150 microliters of 70%ethanol to each tube. Spin the tubes for 10 minutes at 28, 000 times.
G at room temperature. Decant the ethanol air dry, the DNA pellet and resuspend the DNA in an appropriate volume of tris EDTA buffer. This three dimensional reconstruction of a cyber green, one stained viral particle within an agro speed demonstrates how confocal laser microscopy can be used to visualize and confirm that a single virion is obtained in each well after being captured and embedded in aros, the inset shows a higher magnification of the isolated viral particle.
Here a profile plot of the relative fluorescence of the stained single viral particle within an aros bead is shown. This final series of figures shows how almost the entire Lambda genome, with the exception of the first five base pairs, was recovered for this representative virus. Here is the GC plot for the single virus.
Genome identified is shown. This figure shows the genome map of lambda. Finally, the mapping of a single virus genome is shown for the representative isolated and amplified bacteria phage lambda.
The X axis shows the genome position. The Y axis shows the percent coverage. When attempting this procedure, it's important to use sterile technique and to take the time to ensure the contamination is avoided.
Following this procedure, other methods may be used in order to assess the taxonomy of the isolated varians before whole genome sequencing. After watching this video, you should have a good idea how to isolate single varians using fluorescence activated cell sorting, confocal microscopy, and whole genome amplification.
