单缓步动物标本的超低输入基因组测序库制备

The goal of this protocol is to sequence the genome of a microscopic organism called tardigrades. We have established a method to sequence the genome of a tardigrade, Hypsibius dujardini, from a single specimen with as low as 50 picograms of genomic DNA with a whole genome application. After isolating a single tardigrade, we minimize bacterial contamination by using antibiotics and visual inspection.

We have also utilized two homogenization methods. First and most commonly used in C.elegans using freeze and thaw cycles, and second, a manual crushing of the tardigrade with a pipette tip. DNA is then used to construct a sequencing library and then is sequenced in a MiSeq Instrument.

An overall summary of the protocol. After isolation of a single individual, it is subjected to three freeze and thaw cycles for homogenization. The genomic DNA is extracted and purified and is fragmented by sonication.

Then a sequencing library is constructed, and after validation of library size distribution, it is sequenced with a sequencing instrument. Prepare 2%agarose gel using distilled water as the solvent in a 90 millimeter plastic culture dish, and 10 millimeters of 1%penicillin streptomycin with distilled water. The gel can be stored for two to three weeks in an incubator set at 18 degrees.

Collect a single tardigrade and place on the prepared agar plate, and wash with distilled water two to three times to remove remaining particles. Place the single tardigrade in penicillin streptomycin antibiotics for two to six hours to remove bacterial contamination, and place the decontaminated animal on a clean slide glass using a P10 pipette. Observe the tardigrade under a microscope at 500 times magnification, and confirm that there are no remaining bacteria.

Collect individual using a P10 pipette with maximum of five microliters of liquid, and place into a low-binding PCR tube, and remove excess liquid as much as possible. Homogenization and DNA extraction. Homogenize the animal to obtain genomic DNA with one of the following methods.

Homogenization with freeze and thaw cycles. Immediately following step 2.5, add 100 microliters of lysis buffer to the PCR tube containing the tardigrade. Place the PCR tube in liquid nitrogen for 10 minutes, and move to a heat block, warm to 37 degrees for 10 minutes.

Repeat this step three times. Manual crushing. Under a stereo microscope, crush the individual with a P10 pipette tip by pressing the animal against the PCR tube wall, and immediately add 100 microliters of lysis buffer.

Incubate for 30 minutes at room temperature for lysis to occur. Transfer the complete volume of the lysis mixture to a clean 1.5 milliliters low-binding microtube. Add 100 microliters of lysis buffer to the low-binding PCR tube, which is used for homogenization and is now empty.

And after pipetting, transfer the mixture to 1.5 low-binding microtube. Repeat this step twice. Add 300 microliters of lysis buffer to a low-binding PCR tube, and after pipetting, move the mixture to 1.5 milliliter low-binding microtube.

Add the total of 600 microliters lysis mixture to spin column placed in the collection tube, and centrifuge at 10, 000 G for one minute. Reapply the flow through to the column, and centrifuge at 10, 000 G for one minute. This step is critical to ensure that most of the genomic DNA is bound to the column.

Add 500 microliters of wash buffer to the spin column, and centrifuge at 10, 000 G for one minute. Transfer the spin column to a clean 1.5 milliliter microtube. Apply 20 microliters of 10 millimolar first ACL to the spin column and wait for five minutes at room temperature.

Centrifuge at 10, 000 G for one minute. The dilution buffer must not contain EDTA, for it interferes with laboratory preparation enzymes. Reapply the flow through to the spin column, and after five minutes incubation at room temperature, centrifuge for one minute at 10, 000 G.Sequencing library construction.

DNA fragmentation. Transfer 15 microliters of genomic DNA elute To a 15 microliter microtube for DNA fragmentation, and centrifuge for one minute using a tabletop centrifuge. Fragment the genomic DNA to 550 base pairs.

After through pipetting, transfer 10 microliters of fragmented DNA mixture to a clean, low-binding PCR tube. Experiment can be stopped here. Preserve DNA at four or minus 20 degrees.

Sequencing library construction. It is absolutely critical to use the specified kit in the following procedures, due to low-input DNA. Prepare the reagents required, following the manufacturer's protocol, and construct the sequencing library without any modifications.

After applying library amplification mixture to a library synthesis mixture, perform PCR reaction in a thermal cycler. The PCR reaction was conducted as shown. Purification of the PCR reaction.

Add 50 microliters of magnetic beads and pipette 10 times, and centrifuge briefly with a tabletop micro centrifuge. Incubate for two minutes at room temperature. Incubate on magnetic stand for five minutes, or until the solution becomes completely clear, and remove the supernatant.

Follow the manufacturer's protocol. Transfer the supernatant without disturbing the pellet into a new low-binding PCR tube. DNA quality check and quantification, and sequencing.

Validation of DNA library size distribution. Add three microliters of sample water with one microliter of sequencing library, and mix thoroughly for one minute with a vortex, and briefly centrifuge with a tabletop centrifuge. Conduct electrophoresis and validate the library size distribution with associated software.

The main fragment peak should be broadly ranging from about 300 to 1, 000 base pairs. DNA quantification. Add 796 microliters of the solution buffer and four microliters of the fluorescence reagent and mix thoroughly.

Dispense 190 microliters of the working solution to two assay tubes, and 197 microliters to one assay tube. Add 10 microliters of standards with known concentrations of DNA to each assay tube containing 190 microliters of working solution, and three microliters of the prepared library to assay tube containing 197 microliters of working solution. Vortex briefly, and centrifuge on the tabletop centrifuge.

Quantify the DNA using a fluorometer with three microliter settings. Sequencing of the DNA library. Prepare the sequencing library based on the manufacturer's protocol.

Set the reagent cassette and flow cell into the sequencing instrument, and enter the sequencing running information, following the manufacturer's protocol. Run sequencing. Represented results.

Exposure of contaminants in high-quality DNA extraction remains to be the critical point in our protocol. To visually examine if they're contaminant, we have incubated the tardigrade in antibiotics, which are penicillin and streptomycin, and also examined the individual under a 500 times microscope. As shown here, visible microbes around the tardigrades are completely illuminated.

Following efficient homogenization, DNA extraction, fragmentation, and library preparation. The size distribution of the library is validated using a high sensitive electrophoresis for quality control. The purple and green lines indicate upper and lower markers at 1, 500 and 25 base pairs respectively.

Lane L is a ladder marker, lane S and N1 to N4 are five replicates of the same experiment, all starting from a single tardigrade specimen. As it can be seen from the broad uniform and distribution between 200 and 1, 000 base pairs, our protocol is highly reproducible, even with the ultra-low input. Here's the result of fast QC, for synchronized reads obtained from a single sequencing run.

Reads are obtained as spare ends of 300 base pairs, where forward and reverse reads are shown in top and bottom, respectively. The distribution shown here is typical of 300 pair end reads, with very high quality base calling above Q30 for first 200 base pairs, and gradually declining to the end. So this method uses only a single individual for one sample.

There is no requirements of massive amounts of animals, such as those required in previous tardigrade genome sequencing projects. Culturing methods for most tardigrades have not yet been established. Therefore, enabling genomics sequencing from a single individual, such as those directly from the fieldwork, will have a large impact on tardigrade molecular biology, and possibly for other small animals.

Our DNA sequencing methods makes it possible to analyze other numerous microscopic organisms that have not yet been sequenced, such as rare hogweed species, nematodes, water firs, and other options, by connecting the part of the zones and a wider public area, furthering the setting where various biological mechanisms may be possible.