DNA Prep and Sequencing of 5 D. aquaticus isolates, and more qPCR Primer Validation

 Introduction:

This week mostly consisted of prepping DNA samples of Deinococcus aquaticus isolate species that would be used for the first multiplex sequencing run on Illumina technology. These isolates were selected based on environment and previous data collected by another lab, in order to complete the genomes and potentially move forward with a study of the chemotaxis and motility pathways of D. aquaticus. In addition to this DNA prepping, another set of primers for the D. aquaticus qPCR experiment were also validated using identical methodology from the previous run with CSP. By the end of this week, a sequencing attempt was made with three of the five isolates originally planned, and HSP primers were validated. 


Methods:

Five high molecular weight DNA samples, P21, P17, P34, P71, and FR100, were removed from storage in the minus *0 freezer, and allowed to thaw for initial testing of the concentration and purity values to confirm eligibility to be used in DNA prep protocol. The results of these readings on the Nanodrop were insufficent to move directly forward, and DNA cleanup from Monarch was completed using two ethanol washes and a total elution of 20 ul. These samples were then retested on the Nanodrop and proved sufficient to go forward into NEBNext DNA prep for sequencing. The total input for each isolate was approximately 90ng , a volume of 1 ul to 2 ul depending on the sample. Each isolate underwent the entirety of the protocol across two days, with a stopping point after the first bead cleanup step in which they were frozen at minus 20 degrees Celsius overnight. The final 30 ul products were tested using high sensitivity fluorometer reagents and ran on a high sensitivity gel to confirm approximate library size. The found concentrations were used in the Illumina pooling calculator to find 1 nM for each isolate. Two isolates, P34 and P71, were too low in concentration and 1 nM could not be achieved with the given sample and could not be visualized on the gel for sizing. These two were set aside to be redone at a later date and to be multiplexed on a different flow cell. The remaining three, FR100, P17, and P21 were determined to have about 300 bp fragments across the board so were diluted with 10 mM Tris-HCl pH 8.5 accordingly and pooled into a total volume of 20 ul. This sample was directly loaded onto the flow cell and sequencing ran for approximately 18 hours. 

The HSP primer set for heat shock protein was validated with the Luna OneStep qPCR protocol with minor changes but identical to the method of the CSP set. The total input for the first dilution was approximately 80 ng of genomic RNA, and serial dilutions were done in 1:5 with PCR water. Triplicates of five serial dilutions were done, with three NTC control wells and allowed to run on prescribed settings for 45 cycles. The results were extracted and analyzed for primer efficiency calculations to verify primers. 


Results:

The Monarch DNA cleanup kit worked effectively by increasing the purity values well above 2.00 for our samples at absorbance 260/230, and within range of 1.80 - 2.00 for the absorbance at 280/280. There was significant loss of total DNA but multiple attempts at DNA prep for sequencing could be made, which served its purpose. The multiplex run from Illumina appears to be good data, with low contamination values and high coverage values initially being determined, but future processing will validate these findings. 

The HSP primer set was validated with an efficiency of 93.37%, within the range of 90-110%. The third figure shows the calculations made to determine this value with corresponding Cq values from the qPCR run. 


Isolate (Pre-Cleanup)

Concentration (ng/ul)

A260/280

A260/230

P17

418.0

1.95

1.65

P21

660.0

1.97

1.89

P34

430.4

1.96

1.67

P71

417.3

1.96

1.70

FR100

1590.7

1.97

1.98

Isolate (Post Cleanup)

 

 

 

P17

61.3

1.95

2.37

P21

95.8

1.94

2.23

P34

46.4

1.97

2.44

P71

53.7

1.96

2.37

FR100

92.8

1.96

2.29

Isolate (Post Prep)

 

 

 

P17

0.498

 

 

P21

0.551

 

 

P34

0.173

 

 

P71

0.125

 

 

FR100

0.261

 

 

Isolate

Input Solute (ul)

Solvent Volume (ul)

1 nM input (ul)

P17

5.3

8.0

6.6

P21

4.8

8.5

6.7

FR100

10.1

3.2

6.7




Discussion:

This week seemed to be very productive, resulting in the first successful multiplex run from Illumina in the lab and hopefully providing an abundance of data to be used. The DNA prep protocol was analyzed due to the extreme amounts of loss found at the very end of the protocol, which prevented all five isolates from being run and why we had to do just three. It was determined that the prep would change to include a lesser dilution of the adaptor reagent, as this is notated as being experimentally determined in the protocol. The other change will be to do the PCR step with five cycles rather than four, just to boost the total mass as much as possible before going into the final bead purification step. We are hoping with these two modifications that more adaptors will be ligated to the fragments, which will be better recovered in bead purification and will result in an overall increase in final concentration so that no sample will have to be left out of pooling because there is not enough for 1 nM again. Next week five more samples will be attempted, now a combination of isolates and species, including P34 and P71, D. sonorensis, D. metalli, and D. xinjiangensis. D. sonorensis has already been attempted twice, but a new DNA extraction will be carried out to hopefully prevent over fragmentation and have purity to prevent any chances of biological contamination. With the HSP protein being done for the aquaticus primer sets to be validated, the next week will include another one to two sets validated, likely being Cas1 and Cas10.  









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