DNA sequencing analysis - DNA sequencing analysis (Jan/27/2009 )

Put all sequences in the same direction by reverse complementing the reverse sequences, and your alignment looks like this:

CLUSTAL 2.0.10 multiple sequence alignment


seq1 ---------------------------------------AGCTCCTCACTGAGGACTCTG 21
seq1r TTAAAACTTCCATCCAACATCTCAGCATGATGAAACTTTGGCTCCCTACTGGG-ACTCTG 59
seq2 -----------------------------------------AGGAAAAGAGGAAATTCTG 19
seq2r TTAAAACTTCCATCCAACATCTCAGCATGATGAAACTTTGGCTCCCTACTGGG-ACTCTG 59
seq3 -------------------------------------------ACAAACTGTATAATCTG 17
seq3r -TAAAAATTCCATCCAACATCTCAGCATGATGAAACTTTGGCTCCCTACTGGG-ACTCTG 58
* * * ****

seq1 TTTAATCGCACAAATTGTCACAGGGCTATTCCTCGCAATACACTACACGTCTGACATTAC 81
seq1r CTTAATCGCACAAATTGTCACAGGCCTATTCCTCGCAATACACTACACGTCTGACATTAC 119
seq2 TAGGGGGGCACAAATTGAC-CAGGCCTATTCCTCGCAATACACTACACGTCTGACATTAC 78
seq2r CTTAATCGCACAAATTGTCACAGGCCTATTCCTCGCAATACACTACACGTCTGACATTAC 119
seq3 TTAGGGG-CTCAAATTTACACAGGCCTATTCCTCGCAATACACTACACGTCTGACATTAC 76
seq3r CTTAATCGCACAAATTGTCACAGGCCTATTCCTCGCAATACACTACACGTCTGACATTAC 118
* ****** * **** ***********************************

seq1 CACCGCCTTCTCATCCGGAGCCCACATCTGGCGAGACGTCAATTACGGTTGACTAATCCG 141
seq1r CACCGCCTTCTCATCCGTAGCCCACATCTGCCGAGACGTCAATTACGGTTGACTAATCCG 179
seq2 CACCGCCTTCTCATCCGTAGCCCACATCTGCCGAGACGTCAATTACGGTTGACTAATCCG 138
seq2r CACCGCCTTCTCATCCGTAGCCCCCATCTGCCGAGACGTCAATTACGGTTGACTAATCCG 179
seq3 CACCGCCTTCTCATCCGTAGCCCACATCTGCCGAGACGTCAATTACGGTTGACTAATCCG 136
seq3r CACCGCCTTCTCATCCGTAGCCCACATCTGCCGAGACGTCAATTACGGTTGACTAATCCG 178
***************** ***** ****** *****************************

seq1 AAACCTCCACGCCAACGGAGCCTCTTTCTTCTTTATCTGCATTTATTTCCACATCGGACG 201
seq1r AAACCTCCACGCCAACGGAGCCTCTTTTTTCTTTATCTGCATTTATTTCCACATCGGACG 239
seq2 AAACCTCCACGCCAACGGAGCCTCTTTCTTCTTTATCTGCATTTATTTCCACATCGGACG 198
seq2r AAACCTCCACGCCAACGGAGCCTCTTTTTTCTTTATTTGCATTTATTTTCACATCGGACG 239
seq3 AAACCTCCACGCCAACGGAGCCTCTTTCTTCTTTATCTGCATTTATTTCCACATCGGACG 196
seq3r AAACCTCCACGCCAACGGAGCCTCTTTTTTCTTTATCTGCATTTATTTCCACATCGGACG 238
*************************** ******** *********** ***********

seq1 AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 261
seq1r AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 299
seq2 AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 258
seq2r AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 299
seq3 AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 256
seq3r AGGACTATACTACGGCTCCTACCTATATAAAGAAACATGAAACATCGGGGTAATTCTTCT 298
************************************************************

seq1 CCTAC-TAGTTATAATAACCGCCTTTGTAGGTTATGTATTACCCTGAGGACAAATATCAT 320
seq1r CTTAC-TAGTTATAAAA-CCACCTTTGTAGGTATATGTATAAATAACGCC---------- 347
seq2 CCTAC-TAGTTATAATAACCGCCTTTGTAGGTTATGTATTACCCTGAGGACAAATATCAT 317
seq2r CTAAAATAGTTCTTACA-AAAACTCCTGTGATGTAGAGT--------------------- 337
seq3 CCTAC-TAGTTATAATAACCGCCTTTGTAGGTTATGTATTACCCTGAGGACAAATATCAT 315
seq3r CTAAAATAGTTCTTACA-CCATCCCAGCAGGAGAGGAGTTATCCCNNNNC---------- 347
* * ***** * * * * *

seq1 TCTGAGGGGCTGCA----- 334
seq1r -------------------
seq2 TCTGAGGGGCTG------- 329
seq2r -------------------
seq3 TCTGAGGGGCTGCAGTTAA 334
seq3r -------------------

Pretty good agreement -- some minor base calling and end trimming, and you've got your sequence...

Zack,
What this means is that you ether did:
T/A clonilg and half went in as 5' to 3' and the other half went in the other way (3' to 5').
Or you sequenced every thing from both sides.

In any case, you have the same sequence every time.