Part 1: Opening your sequence with FINCH and evaluating its quality:

Put the following sequences in order of quality.

Best _________ ________ __________ _________ Worst

A B

C D

Part 2: In class we will use FINCH TV, but here use the text version of these
sequences to find and remove all the parts from the plasmid.

Below are two full length sequences. Go through those sequences and highlight the
parts of the DNA below that you would remove. For all of your reactions the plasmid
sequences. AATTCGCCCTT (to the left) and AGGGCGAATTC (to the right) flank either side
of the PCR product.

NOTE: you need to find the listed sequences in your DNA. However, the beginning of the
DNA sequence often has N’s, and if the sequence you are looking for is right at the
beginning it can be difficult to locate because some of the bases you are looking for may
be replaced with those N’s. If you can’t find the full length sequence try searching for
smaller sections till you do find something (for example, for the left hand sequence, you
can search for TCGC, and then identify the rest of the sequence you are searching for).
Then look to make sure that the sequence with the N’s replaced would actually match
your sequence.
When you are done you should check if you have done this correctly by comparing
your remaining sequence with the similarly labeled sequence from the
“Comparison” File in Part 3

DNA #1
>NT-9-3-T7_H05.ab1
NNNNNNNNNNNGNCNCNNTTCGCCCTTAATGTGGTATTGAAGCTAAGTATGGATTAGT
TTTTTCCGAGTTTGGTGTTGGATATTTTTGTGGATATATGGTCGATTACGATGAGGTTG
GTATTTTTGTTTTTGTATTATTTGTAATGTTATAGCGGTATTTATTATTTTTTTGTAGG
AGACGAGGAAATGCAGTTGGGAGAGTTACGACGATAAAGGAGAGGTGATGCGTATAATA
GAGTTTAAGAGAATAATGGGATAAAAGATGTATTGTGGATTTCGGTTCGTCGATTCGTA
CATTAACGATTGTATACGCGATATGCGTAGGGAAGTAAAATCTGTTTTTTATGATAGTC
CGAAGCGTGCAGAGTGTAGGAGGTTGAAGGCGGTAGATGAGAAGAGAGAAGGGCGAATT
CGTTTAAACCTGCAGGACTAGTCCCTTTAGTGAGGGTTAATTCTGAGCTTGGCGTAATCA
TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA
GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT
TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG
AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCT
CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC
GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAG
GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC
GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCANANTNNN

DNA #2
>T-1-4-T7_D04.ab1
NNNNNNNNNNNNNNNNNNNTTCGCNNTTNNGAGGAGAGTGTTTTAAGGTGGGGTTT
GAAATTTGGTATTAAAAAGATTGAAAGAGTTTTTTATTTTATTATATGTTTATTTGAGT
TATAAAATAATTGATTTTGTTAATTAAATTGTAATAATATATGGGTATTTGGTTAGTAT
AGTTTTAATGGTGGGAGAACGATAAGAGGAAATTTTGTTTTTTTATGAATAATTTAATG
TGTTTTAAATATTTTTAATTTTTGGAATTAAATATGTTAGAGATTAAATTTTAGATGTT
AGATTAAATATGGTAGTTTTATAAAGATAAATTTTTGTATAGTTTTTAAATTAAGTTAT
TAGAGTTTTTTTAAGAAATATTATATTAAATATATTAAATAAAAATGTTTGTCGGTGTG
TCAGGAAAAGGGCGAATTCGTTTAAACCTGCAGGACTAGTCCCTTTAGTGAGGGTTAAT
TCTGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC
AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG
TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT
CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT
ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA
AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG
GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG
ANGTGGCGAAACCCGACAGGACTATAAAGATNCCANGGCN
Part 3: Aligning and Analyzing

Below is a picture of an alignment from Mega.

The top sequence is the reference sequence which was not bisulfite treated.
 Put and arrow pointing to all C’s that are mostly methylated,
 Put a Star above C’s that are mostly unmethylated.
 Circle individual C’s that do not match the methylation state of the C’s at that
position in other sequences.

Next we will have you analyze an provided alignment file in CyMate.

https://www.cymate.org/

You should be able to put these files in and get out the overall statistics, and the dot
plot.

List those results here:

Total Methylated Total Unmethylated Total

CG
CHG
CHH
ALL

Looking at the Dot Plot:

Are there any C’s that are methylated only under one condition or the other?
Describe the pattern that you see for each of these C’s. What might this tell you
about these C’s and the treatment?

Which condition shows more methylation?

Are there any C’s that are consistently methylated regardless of the condition?