Preparing 5 Samples for Multiplex Sequencing and More Primer Validations

 Introduction:

This week there was a continuation of sequencing, as three isolates were successfully completed given the current data analysis. The two isolates that were not in sufficient mass to reach a 1 nM concentration for loading, P34 and P71, were reprocessed again with the adjustments made to the protocol including an additional PCR cycle and not diluting the adaptor solution added to the samples. A DNA extraction had to be completed for D. sonorensis due to contamination suspicions the last time it was attempted, and this new sample was processed making up the third specie included in this run. Samples of D. metalli and D. xinjiangensis that were DNA extracted approximately a year and a half ago were cleaned up and accounted for samples four and five for the pooling and sequencing attempt. Finally, SecA and Gap3 reference genes were primer validated and results were calculated for efficiency. 

Methods:

D. sonorensis was DNA extracted by growing cells on R2A plates for 72 hours and scraping an unspecified number of colonies from plate and directly into R2B media and homogenized by vortexing. Note, this was tried with cells directly into water and the cells did not pellet in later steps for the DNEasy cleanup kit. The R2B samples were pelleted properly, and underwent the kit with slight modifications. One sample was intended to be bead beated at max speed (5m/s) for two minutes and the other 5 for minutes (1 min on/1 min off on ice) but the resulting lysate was accidentally combined so both samples were 50% 2 minute bead beating and 50% 5 minute bead beating. They were each eluted in 30 ul of nuclease free water with an incubation at ambient temperature for about 5 minutes prior to centrifugation. The results were recorded using a Nanodrop One and listed in Figure 1 of results. 

D. metalli and D. xinjiangensis samples were removed from storage and tested for cleanliness using the Nanodrop One. When determined unfit for sequencing, they each underwent the Monarch DNA cleanup kit protocol, with an additional incubation of about five minutes prior to centrifugation of the eltuion volume, which was 12 ul each. Results were again tested using the Nanodrop One, and stored at minus 20 degrees Celsius for late use. 

DNA prep was carried out per the protocol for the five samples. P34, P71, sonorensis, metalli, and xinjiangensis each with an approximate input of 85 ng. The modifications mentioned last week were applied, with lack of dilution of the adaptor solution for adaptor ligation and an addition of the fifth PCR cycle later in the protocol. The samples were ran on a high sensitivity gel to verify average fragment size, and tested on a fluorometer for concentration. The Illumina pooling calculator was used to determine 1 nM concentration dilution values for sequencing with a total 20 ul sample. 

Primer validation for Gap3 and SecA was completed using Luna OneStep qPCR protocol with a starting input of approximately 66 ng in mass, with 1:5 dilutions thereafter. Triplicates of each dilution was done, as well as duplicates of NTC wells for each primer set.  

Results:

The primer efficiency of SecA could only be determined since Gap3 was unusable data with detection at cycle 29. The efficiency calculated from the given Cq values was 94.6%, well within range and so these primers are considered validated. 

The results of the sonorensis DNA extraction were in high concentrations, at 323.3 and 388.0 ng/ul each, but it is undetermined whether or not how reliant these results were on the 5 minute bead beating versus the 2 minute bead beating sample. The cleaning up of D. metalli and D. xinjiangensis was also very successful, with minimal loss of DNA and a significant increase in cleanliness values that allowed each to be included on the Illumina sequencing run, surpassing parameters. 

The Illumina sequencing run has not been detailed as being successful or not as of yet. 

Sample

Concentration (ng/ul)

A260/A280

A260/A230

D. sonorensis (1)

323.3

1.94

2.12

D. sonorensis (2)

388.0

1.95

2.13

 

 

 

 

D. metalli (before)

51.8

1.84

1.65

D. xinjiangensis (before)

95.9

1.88

1.93

 

 

 

 

D. metalli (after)

59.4

1.91

2.13

D. xinjiangensis (after)

264.8

1.93

2.21

 

Sample

Concentration (ng/ul)

Input Solute (ul)

Solvent Volume (ul)

1 nM input (ul)

P34

2.57

2

29.2

4

P71

2.67

2

30.4

4

D. sonorensis

3.12

2

35.8

4

D. metalli

2.84

2

32.4

4

D. xinjiangensis

2.65

2

30.1

4

 




Discussion:

This week yielded high productivity for the most part in the lab. The Gap3 and SecA primer validations at least halfway worked, and the Gap3 primers will need to be evaluated for either human error or a possible secondary binding site according to the Melt curve that was produced. Hopefully this is human error and can be fixed next week with more careful pipetting, as this would set back the experiment being a primer reference gene. The DNA extraction of D. sonorensis was very exciting, to see that less bead beating could be used to get objectively great results likely due to the fact that it was grown on R2A to begin with which allowed for homogenization. It is still extremely interesting that the cells would not properly pellet unless put into R2B, and when put in water it was impossible to get a solid pellet without a strange oily swirl throughout the whole tube. This phenomena should be documented and looked at later. It was also encouraging to see that the changes made in the DNA prep protocol for Illumina made a significant difference in yield at the end of the protocol, across the board of five samples. It is yet to be seen if these sequences will be usable, but it seems the protocol for preparing DNA has been better optimized for our species. 










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