DNA Prep for Illumina Sequencing

Introduction:

Last week the preparation was done to be able to sequence five samples using Illumina short read technology. The list of samples includes three species, two of which have not been sequenced in the lab before, and the two isolates of Deinococcus aquaticus. This week, D. pimensis, D. grandis, D. caeni, P81, and P65 were all prepared and sequenced. These sequences are so critical because they are provide new insight into each of these samples that can be utilized for potential experiments.  


Methods:

The samples were thawed and centrifuged, with approximately 85 ng of total mass used as input for the Illumina protocol. The samples were diluted with 1X TE provided by the kit to meet the volume requirement for the fragmentation step, which had zero modifications to it. Post fragmentation, adaptors were ligated with zero adaptor dilution as per experimentally validated protocol. The samples were cleaned using the magnetic beads, and eluted in 15 ul of 0.1X TE Buffer solution and stored in the minus 20 degrees Celsius overnight. The next day, the samples were again thawed and centrifuged, and the entirety of the volume was input for the barcoding and subsequent PCR enrichment step with a modification to have 5 cycles rather than 4 as was used previously with lower yields found at the end of the protocol. The samples were finalized by cleaning suing magnetic beads again, and eluted in 30 ul of 0.1XTE Buffer. The samples were measured using 2 ul on the high sensitivity fluorometer and readings determined the amount of volume used for the gel. The gel ran was a 2% gel using GelRed agarose, in which every sample was 3 ul of input with the exception of P65, which had 6ul, for a total of approximately 10 ng in each lane. A 50 bp ladder was used to measure the average size of each sample post preparation, which would be used to determine the sample dilutions for the 1 nM pooling of all 5 samples. They were stored in the minus 20 degrees Celsius for another overnight, and their pooling calculations done the next day. They were pooled in a total volume of 20 ul, and then diluted to 100 pM by a 2:18 dilution using 10 mM Tris-HCl pH 8.5 for both the pooling and dilution steps. The flow cell was removed form the 4 degrees Celsius fridge and allowed to equilibrate to room temperature for 20 minutes before being inverted 5 times, gently hit against the tabletop 5 times, and then loaded with the final 20 ul of 100 pM pooled samples. The run was started successfully and allowed to go for about 18 hours. 


Results:


Sample

Fluorometer (ng/ul)

Loaded on Gel (ul)

Volume for pooling dilution (ul)

Volume for solvent in pooling dilution (ul)

Volume from each dilution into 20 ul pooled sample

D. pimensis

3.51

3

2

51.2

4

D. grandis

4.15

3

2

60.9

4

D. caeni

3.64

3

2

53.2

4

P65

1.63

6

2

22.7

4

P81

3.22

3

2

46.8

4





Discussion:

The samples seemed to be prepared well, all with sufficient results off the of the fluorometer readings. P65 being about half as much as the others was slightly interesting, may of been due to unfortunate diluting or a lack of proper mixing at various steps during the protocol. There was a point in time during the air drying of the magnetic beads the second time that some of the tubes may have been overdried and that may have been the case for  P65, which is why it had less yield. Thankfully, there was still plenty of sample to be able to do a 1 nM dilution with pooling, so it was not an issue. The gel was also interesting because the image does not show the clarity which was actually seen, but the smears maybe should have been more prominent with the amount of mass loaded into each well. This new dye is still be worked out, and maybe 10 ng of mass should have been increased for some better resolution, but it seemed to have been okay. The Illumina sequencing run was ultimately a failed attempt, but it seems it was a technical issue as the run got to the 100th cycle and then glitched with barcodes reading incorrectly, and it wad deemed technology rather than a biology issue so the flow cell will be replaced and these samples will be run again and hopefully success the second time around. Next week most of the time will be dedicated to finalizing poster presentations to prepare for the conferences next week. I will discuss my poster and the findings that we will be presenting with D. sonorensis. 

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