During this lab we worked on reference based and de novo assembling. We examined the DNA sequences of various organisms and utilized tools found in Geneious to accurately assemble their DNA sequences. We also scored our ISSR analysis gels. These scorings will assist with comparisons later.

Reference Based Assembly questions:

1. 4911, 4909, 4871, 497, 472,
2. De Novo because paired-ends help researchers when there are no reference sequences. Although they can be used in reference sequences, the reference itself is a clue, whereas in de novo that clue is unavailable.
3. 5.28 seconds
4. average coverage: 98.1, max: 139. Lowest coverage came from the ends of the strands. This is probably because it’s harder to multiple fragments that line closer to the tips of the consensus sequence.
5. 38,20,259,234. These sites are highly polymorphic
6. 34->47, excluded because low coverage, invoking less confidence
7. transitions: 4575,4567 transversions: 311, 4497
8. OK
9. 4499->4582 14 excluded.   293->310 none excluded
10. 
11. 25,172 reads, 4 contigs, 17994,511,67096
12. 41.7 seconds. Contig 4 low, contig 1 highest
13. 90759->90774 & 92078->92266
14. 24.4 seconds
15. The consensus contained ambiguous bases when we would prefer mode bases. 58,851, 83786 & 268247
16. 269124 bp.

ISSR Scores

I referred to the short runs initially for bands and looked at the long runs to determine if there may have been any potential overlap. I also excluded individuals without both markers.

Protocol:

1. Added 1 microliter of loading dye to ISSR PCR products
2. Loaded onto gel for imaging
3. ran at 60 volts for about an hour and a half

Analysis of Lupinus ITS Sequences

1. Downloaded Lupine_MEC_2017 file from Canvas
2. Opened file in geneious
3. Trimmed then assembled forward and reverse DNA sequences
4. Selected all consensus sequences and made an alignment using default parameters of Muscle.

Results:

Write Up Questions

1. Yes, my tree had one clade with a support value above .85. They were somewhat close together, and they all seemed to be from populations that had yellow flowers.
2. Yes, a few populations that had purple flowers belonged to the same clade as other purple-flowered populations
3. I believe that although ITS sequences can distinguish populations somewhat, the differences in these sequences are not enough to draw solid conclusions phylogenetically. This is because a lot of sequences failed to be grouped into distinct clades with other sequences. As a result of this, it is hard to claim that the ITS is useful enough to group these differing color populations phylogenically.

Protocol

1. Added 1 microliter of loading dye to PCR products
2. Ran PCR products in 1.3x agarose loading gel
3. Ran for about 1 and a half hours at 60v
4. Imaged gels to determine which ISSR’s work best
   1. Decided to use 17898 and OMAR
5. Made 1:10 dilution of Plant DNA (10 microliters DNA, 90 Microliters water) from GWC samples
6. Made two master mixes with
   1. ddh20        12.5 microliters
   2. 10x buffer + MH     3 microliters
   3. BSA            1 microliter
   4. dNTPs        2 microliters
   5. Primers     .25 microliters (either OMAR or 17898)
   6. tax              .25 microliters
   7. template    1 microliter
7. Ran on thermocycler