DNA Preparation for Further Illumina Sequencing

 Introduction:

In previous weeks Illumina sequencing has been well underway, with multiple species and some isolates have newly developed de novo sequences that has provided a plethora of information and insight. This week was a continuation and preparation for the following week in which more isolates and species will be sequenced, including a replication of D. caeni due to incomplete data acquired the first run on Illumina. The two isolates chosen are considered to be 16s outliers in comparison to other isolates, and suspected of being the most genetically variant from type strain of D. aquaticus, PB314. The other two species, D. pimensis and D. grandis were chosen due to only having limited sequencing data available. D. pimensis has only scaffolding information and D. grandis has contig files is listed as having high levels of contamination that can and should be improved upon for clarity. This week included ensuring the quality of the stored DNA samples, cleaning up a few of them that were insufficient, and calculating necessary dilutions to prepare for the Illumina DNA Prep protocol. Next week will include the actual prep, as well as sequencing, and the beginning of preparations for ONT sequencing that will provide long read data to create hybrid sequences with the recently collected Illumina data. 


Methods:

The samples were collected from the -80 degrees Celsius freezer, thawed, and measured initially on the Nanodrop One. Once tested, D. grandis and D. aquaticus P81 were processed through the Monarch NEB DNA cleanup and purification kit, with a 2:1 binding buffer ratio to select for larger fragments and then a five minute ambient incubation prior to the final centrifugation with 10 ul of PCR water to elute. The samples were tested again on the Nanodrop one to be confirmed for approximate concentration and cleanliness. All samples were diluted using provided 1X TE Buffer from the Illumina DNA processing kit, with the given values below in Fig. 3. Samples were again tested using the high sensitivity fluorometer setting and recorded. The necessary values for 85 ng of total DNA mass input were also calculated. 

The Illumina DNA prep was originally to begin this week, but confusion surrounding the volumes of reagents left in the kit temporarily delayed the progress and processing will begin the following Monday with these calculated values. Samples were stored in the -20 degrees Celsius freezer. 

D. sonorensis plates were inoculated on 3:3:1 TGY from freezeback to prepare for imaging the following week. Another set was inoculated the following day when no growth was seen on the first two plates after 24 hours. A second passage was inoculated on the third day to provide for the freshest cells on Monday morning. 


Results:


Species

Concentration (ng/ul)

A260/A2380

A260/A230

D. aquaticus P65A

56.0

1.95

0.86

D. aquaticus P65B

205.1

1.92

2.06

D. aquaticus P81

99.6

1.98

1.09

D. pimensis 1

550.0

1.96

2.13

D. pimensis 2

621.1

1.92

2.13

D. caeni

550.4

1.96

2.12

D. grandis

25.0

1.98

1.79

Fig. 1: Initial Nanodrop One readings from samples directly from the -80 degrees Celsius storage. 


Species

Concentration (ng/ul)

A260/A2380

A260/A230

D. grandis

56.8

1.94

2.01

D. aquaticus P81

166.4

1.93

2.06

Fig. 2: Nanodrop One readings post DNA cleanup and concentration in 10 ul of total elution volume. 


Species

DNA Sample (ul)

1X TE Buffer (ul)

Total Volume (ul)

D. grandis

2.277

12.723

15

D. pimensis 1

0.309

19.691

20

D. caeni

0.309

19.691

20

D. aquaticus P65B

0.829

19.171

20

D. aquaticus P81

1.022

18.978

20

Fig. 3: Calculated dilutions using C1V1=C2V2 for sample concentrations to be an approximate 8.5 ng/ul. 


Species

Concentration (ng/ul)

Volume for 85 ng total mass (ul)

D. aquaticus P81

6.09

13.96

D. aquaticus P65B

6.47

13.14

D. caeni

7.48

11.36

D. pimensis 1

8.82

9.64

D. grandis

8.76

9.70

Fig. 4: Fluorometer readings and calculated volumes necessary for 85 ng total input for each sample for the Illumina DNA Prep protocol. 


Discussion:

A lot of prep was accomplished this week in order to provide a smooth start for the following week, which should be very busy concerning lots of DNA prep for both Illumina and ONT samples. The hope is that D. sonorensis grows well over the weekend from freezeback to provide ample cells to fix and deliver to ASU for both SEM and TEM imaging.  




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