DNA Sequencing and Extraction of Deinococcus caeni

Introduction:

Nanopore sequencing with Deinococcus caeni has been the goal for the past week or so in the lab, but this week it came to fruition unsuccessfully. The DNA extracted last week was processed in the Nanopore Ligation V14 kit and sequenced attempted, and then post sequencing sample analysis of approximate genome size for more in depth analysis in preparation for the next sequencing attempt. 


Methods:

The first step in preparing for sequencing was to do a DNA cleanup and concentration of the sample produced from the previous week. The NEB Monarch DNA cleanup kit was utilized by diluting the 18 ul of sample to a total volume of 50 ul with a 1:2 binding buffer ratio to select for genomic DNA at higher kbp fragments. The protocol was followed until the elution step in which  15 ul of PCR water was used as the eluent and an incubation step at room temperature for five minutes prior to centrifugation. The sample was tested for concentration and cleanliness on the Nanopore One and then verified with the Broad Range QUBIT Fluorometer for a specific concentration value. 

The Nanopore Ligation V14 included an initial input of approximately 1 ug of DNA template from the sample previously cleaned up. Then 6.6 ul of sample was diluted with 40.4 ul of water to bring the 1 ug of DNA to a total volume of 47 ul required for the protocol. The protocol was followed, and the optional addition of DNA CS (control sequence) was used. Hula mixing was done at 120 RPM for the designated 5 minutes at room temperature. The High Sensitivity QUBIT Fluorometer reading was done after the fist day of DNA prep involving end preparation for adaptors using 1 ul of sample and then the remaining 60 ul was stored at 4 degrees Celsius overnight. 

The sample preparation was continued the following day, which included adaptor ligation and bead cleanup with the specific short fragment buffer for all fragments to be carried into the DNA elution to ensure high enough DNA concentration since the Long fragment buffer will only select for fragments grater than 3 kbp. The sample was eluted and another High sensitivity fluorometer reading was completed and the sample was calculated and diluted to 20 fmol assuming a 2 kbp average fragment size. This was a 26.64 ng total loading mass based off of these calculations, using 1.34 ul of sample and 13.66 ul of elution buffer to be able to load 12 ul for sequencing. The Nanopore flow cell was loaded and allowed to run for 12 hours, with initially 1400 pores detected but due to loading issues was down to about 500 pores at the start of sequencing. 

The original DNA sample of D. caeni from cleanup was assumed to be 2 kbp in average fragment length but 2 ul of run on a GelRed 1% gel with a 1 kbp ladder to properly evaluate average length. Another set of DNA extractions using the DNEasy kit was done, with 24 hour TGY broth culture approximately 20 ml. A gram stain was completed to ensure lack of contamination, and then 4.8 ml of culture was used for each sample. One was done at 5 m/s bead beating power for 15 seconds, and the other was 4 m/s for 30 seconds, in an attempt to evaluate any change of average base pair size for future sequencing. Both samples were eluted in 20 ul of elution buffer after a 5 minute incubation at room temperature prior to centrifugation. They were both tested on the Nanodrop One for cleanliness and concentration. 

Results:

The Nanopore sequencing was unsuccessful with only a total of 36 reads actually done which all failed, but this may have been expected since the integrity of flow cell was knowingly compromised from the beginning. 

Sample

Concentration (ng/ul)

A260/A280

A260/A230

4 m/s 30 sec

161.3

1.92

2.03

5 m/s 15 sec

136.1

1.92

1.92

 

 

 

 

DNA post cleanup

153.6

1.94

2.14

Post cleanup QUBIT

152.0

 

 

 

 

 

 

Day One QUBIT

12.6

 

 

Final Elution QUBIT

23.0

 

 



Discussion:

The Nanopore sequencing was a failed attempt but this was likely due to the compromised flow cell, and potentially not helped by the average base pair size of the fragments being off which would have changed the actual fmol loaded into the sequencer. Next week will include another gel of the newly extracted D. caeni samples to compare to this one and hopefully see a change in average fragment length that can be more optimal for ligation sequencing, which prefers high molecular weight DNA. 





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