Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

This video demonstrates the use of an automated gel size selection instrument to achieve improved reads in next generation sequencing platforms. First environmental water samples are collected and passed through a series of filters to systematically separate eukaryotic bacterial and viral sized particles. The retained bacteria are further concentrated by centrifugation.

Then the bacterial DNA is extracted. The extracted DNA is used to prepare libraries in which adapter and indexes are incorporated into fragmented DNA. The DNA is then amplified by PCR, and samples are loaded into the electrophoresis workstation.

DNA fragments within a specific range are then automatically selected. Analysis of traces from chip based capillary electrophoresis and additional bioinformatics demonstrates that this method yields suitable quantities of DNA fragments for downstream analysis with minimal clustering of unwanted fragments under 200 base pairs. The main advantage of the automated Electrophoresis square station or paramagnetic beats and other commercial systems is that it allows for a tighter, narrower range of selection in which up to 96 samples can be processed in a single run.

Though this method can provide insight into microbial communities in freshwater samples, it can be applied to other systems such as seawater, wastewater treatment, plant samples, sediment soil, or microbial mats, essentially any DNA sample for which we want a narrow selection of sizes. We first had the idea for this method when we saw short fragments in MySEQ platform sequences. We tried SI several size selection approaches, including different types of para beads and ratios, as well as manual gel size selection approach.

Then we started to collaborate with Coastal Genomics, a privately owned British Columbia Corporation offering the Ranger technology Demonstrating the procedure. Today will be Michael Chan, research technician from my laboratory, Miguel Yi, a postdoctoral fellow from my laboratory, and Jared Sloan, an engineer from Coastal Genomics. In this video, we will only describe the purification and analysis of the material recover on the 0.2 micron filters, primarily free living bacteria, but keep in mind that similar approaches can be used for the other particles recovered from the filters.

Begin this procedure by collecting 40 liters of freshwater samples from several environmental sites in sanitized 20 liter carboys. Pre-filter the samples in situ using a 105 micrometer spectrum mesh polypropylene filter. Here samples are collected from urban and agriculturally influenced watersheds.

Following collection, place the samples in coolers and transport them back to the lab there. Store the samples at four degrees Celsius. The next day.

Set up a filtration system that consists of the sample collection carboy connected via a peristaltic pump to an enviro check one micron sampling capsule. Connect the sampling capsule to a 0.2 micron disc filter and connect that to a 30 kilodalton tangential flow filter By way of a second peristaltic pump. Turn on the peristaltic pumps to pass the samples through a series of filters, the eukaryotic cells will be isolated as they pass through the sampling capsule.

The bacterial fraction will be isolated on the membrane disc filter, and the viral particles will be isolated on the tangential flow filter. Remove the 0.2 micrometer membrane filter or filters from the water filtration system. Then fold the filter in half and place it into a 50 milliliter tube.

With the open side up. Add five milliliters of PBS with tween to the tube. If more than one filter was used during the filtration, use a different tube for each filter.

The filters can then be stored at four degrees Celsius or processed right away as follows. Next, using sterile forceps, remove the membrane filter from the 50 milliliter tube and place it on a Petri dish with sterile forceps and sterile scissors. Cut the filter into one centimeter by one centimeter pieces between samples.

Disinfect the instruments by soaking them in 70%isopropanol, wiping them down with a DNA surface decontaminate, and then rinsing them with type one ultrapure water. Place the filter pieces back into the same 50 milliliter tube. Add 10 milliliters of PBS with tween to bring the total volume of buffer to 15 milliliters using sterile forceps.

Add six clean tungsten beads, three millimeters in diameter to each 50 milliliter tube. Cover the tube with paraform and vortex. It vigorously for 20 minutes.

After vortexing pool all suspensions in a single clean 50 milliliter tube for each sample. Centrifuge the tubes at 3, 300 GS for 15 minutes at four degrees Celsius. After the spin, discard the supernatant and resuspend the pellet.

In five milliliters of PBS solution, distribute the bacterial cells in one milliliter aliquots into 1.7 milliliter micro centrifuge tubes spin in a micro centrifuge at 10, 000 GS for 10 minutes. Following the spin, use a pipette to remove all but 200 microliters of the supernatant. Store the samples at negative 80 degrees Celsius or proceed to extract bacterial DNA using a commercially available DNA isolation kit.

During bacterial DNA extraction, use either PBS or water as a negative extraction control and e coli as a positive extraction control. Following the DNA extraction procedure, pool the aliquots from the same sample into one two milliliter tube in 10 millimolar tris buffer. Then conduct an overnight precipitation by adding a solution comprised of 10%three molar sodium acetate, two volumes of 100%ethanol and five microliters of linear acrylamide Store samples at negative 80 degrees Celsius.

The next day, centrifuge the samples at 17, 000 GS for 30 minutes at four degrees Celsius. After the spin, wash the pellet with one milliliter of ice cold. 70%ethanol allow the pellet to air dry in a biosafety cabinet for no more than five minutes.

Then resuspend the DNA pellet in 10 millimolar tris. CL finally determine the DNA quantity and quality by performing a high sensitivity fluorescent nucleic acid quantification assay, followed by micro volume spectrometry. After determining the quantity of DNA in each sample, add the appropriate amount of water to each tube to bring the concentration to about 0.2 nanograms per microliter.

Using a commercially available kit, prepare a DNA library of indexed fragments from one nanogram of bacterial DNA. Here, a transpose on based method known as mentation is applied to simultaneously fragment in corporate adapter sequences onto DNA. Then PCR is performed to simultaneously amplify and index the ED samples, thereby providing specific barcodes on each fragment and sequences required for cluster formation that can be read by the sequencing platform.

Following completion of PCR, skip the standard post PCR cleanup step and add 3.5 microliters of loading buffer to each sample for a total volume of 28.5 microliters. Then transfer the samples to a 96 well plate load. The 96 well plate into an electrophoresis workstation with a precast 1.5%agarose cassette in the Ranger software user interface.

Specify a size selection target of 500 to 800 base pairs. Set the extraction volume to 300 microliters. Initiate the run.

The electrophoresis workstation will automatically load the samples into the aros cassettes. Then voltage is applied to the cassettes, and a gantry mounted imaging system is used to identify the location of sample DNA in the aros. Throughout the run.

The Ranger software captures images and using internal standards estimates the position of the size selected target in each sample. After the targets are identified, the ranger software varies. The voltage applied to each sample to synchronize the arrival of the targets at the cassette extraction wells.

Finally, the electrophoresis workstation aspirates the targets in TBE from the extraction wells and dispenses them in a new 96 well plate to concentrate the raw output material, precipitate the raw elucian volume using sodium acetate and ethanol with linear acrylamide overnight at negative 80 degrees Celsius. Centrifuge the samples again at 17, 000 GS for 30 minutes. After the spin, discard the supernatants and proceed to wash the DNA palate with one milliliter of ice cold, 70%ethanol centrifuge.

The samples at 17, 000 GS for 30 minutes, discard the supernat air, dry them, and finally resuspend the DNA pellet in 10 millimolar tris CL proceed to normalize samples using library normalization beads, which are included in the transpose on base library preparation kit. Finally proceed with DNA sequencing using a next generation sequencing platform to yield more informative reads in next generation sequencing platforms. By improving the quality of sequencing libraries.

Water samples were taken at eight different watershed sites and index DNA libraries were prepared from the isolated bacterial fraction as shown in this electropherogram DNA libraries generated from freshwater samples following PCR, but prior to automated size selection were comprised of fragments ranging in size from 50 base pairs up to 3000 base pairs. Following size selection using the high throughput automated platform, the samples a tight range of DNA fragments between 500 and 800 base pairs. Thus, utilization of this automated gel size selection platform resulted in suitable yields and minimized the clustering of unwanted fragments under 200 base pairs.

DNA sequencing of this library on the aluminum MiSeq generated a cluster density of 1198 K per square millimeter. The percentage of cluster passing filters was 84.2%with an estimated yield of 9.2 gigabytes. Moreover, 7.4 gigabytes, or 82.8%of the reads had a quality score of greater than or equal to 30.

This means that approximately 83%of the basis sequenced with this modified protocol and applied to environmental water samples had a base call accuracy of at least 99.9%After watching this video, you should have a good understanding of how to generate more informative reach from sequencing libraries When performing this procedure is important to remember the microbial fraction you are aiming for the choice of library preparation, the target fragment length, and sequencing platforms.