Sanger sequencing

Sanger sequencing

Brief chromatogram description

Non-readable regions:
Direct sequence Reverse sequence
5' 1-23 1-2
3' 705-719 687-718

In general, both chromatograms look okay. Few mistakes, ratio of data peaks to noise peaks is about 10.
Reverse sequence is a bit longer that the direct one: T54 from the reverse equals to T2 from the direct reading.
Figure 1. T1 and T54 in direct and reverse sequences.

I've also found two unusual regions:


Figure 2. Unusually low signal
(direct sequence)
Figure 3. Dye blob
(reverse sequence)

Jalview alignment


 
file1/1-681file2/1-684
102030405060708090100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400410420430440450460470480490500510520530540550560570580590600610620630640650660670680690700710720730-----------------------------------------------------TTTTATATTTGGCGCCTGAGCAGGTACAGTAGGGACTGCCATGAGAAAAATTATACGAGTTGAACTTTCTCAGCCAGGCTCTTTAATACAAGATGATCAAGTATATAAaGTTATGGTAACGGCCCACGCCTTCGTCATGATATTTTTTATGGTAATGCCCATAATGATAGGGGGGTTTGGCAAATGACTTGTCCCACTAATGTTAGGAGCGCCkGATATGGCTTTCCCCCGAATGAAAAAAATGAGATTTTGGCTACTACCCCCAGCTTTTATACTTCTTCTAGCTTCAGCTGCAAACGAAGGAGGAGTAGGCACTGGATGAACTATTTATCCCCCTTTGtCAGGCCCTACCGCACATGCAGgAGGCTGCGTAGACCTCGCAATTTTTTCTCTCCACCTAGCAGGTGCGTCTTCAATTATGGCCTCAATAAAATTTATTACAACTATwATAAATATGCGTAGGCCCGGCATGACCATGGATCGACTTCCACTTTTTGCTTGATCTATTTTCTTAACAACTATATTACTACTCCTTTCTCTGCCTGTTTTAGCAGGAGCTATTACAATGCTATTAACTGATCGTAACATAAAAACAACGTTTTTTGATCCTACAGGAGGAGGAGACCCAATACTTTTCCAACATTtATTTTGrTTyTTyGGCCAyCCCGAGGtCTAGTCATATTGTAAAACGACGGCCAGTTGTCGACGAATCACAArGAhhTTGGAACACTATATTTTATATTTGGCGCCTGAGCAGGTACAGTAGGGACTGCCATGAGAAAAATTATACGAGTyGAACTTTCTCAGCCAGGCTCTTTAATACAAGAyGATCAAGTATATAAAGTTATGGTAACGGCCCACGCCTTyGTCaTGATATTTTTTATGGTAATGCCCATAATGATAGGGGGGTTTGGCAAATGACTTGTCCCACTAATGTTAGGAGCGCCTGATATGGCTTTCCCCCGAATGAAAAAAATGAGATTTTGGCTACTACCCCCAGCTTtTATACTTCTTCTAGCTTCAGCTGCAAACGAAGGAGGAGTAGGCACTGGATGAmCTATTTATCCCCCTTTGTCAGGCCCTACCGCACATGCAGGAGGCTGCGTAGACCTCGCAATTTTTTCTCTCCmCCTAGCAGGTGCGTCTTCAATTATGGCCTCAATAAAATTTAyTACAACTATTATAAATATGCGTAGGCCCGGCATGACCATGGATCGACTTCCACTTTTTGCTTGATCTATTTTCTTAACAACTATATTACTACTCCTTTCTCTGCCTGTTTTAGCAGGAGCTATTACAATGCTATTAACTGATCgTAACATAAAAACAACGTTTTTTGATCCTACAGGAGGAGGaGACCCAATA--------------------------------------------------

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My edits

# 1 2 3 4 and 5
Direct sequence
Figure 4a.
Figure 5a.
Figure 6a.
Figure 7a.
Reverse sequence
Figure 4b.
Figure 5b.
Figure 6b.
Figure 7b.
Description Unidentified nucleotide (N #341) was replaced to T (according to thymine #394 in the reverse sequence) N #364 in direct replaced to G (see reverse guanine #416) N #110 in direct replaced to A (see reverse adenine #162) N #114 in reverse replaced to T (see direct thymine #62)
N #147 in reverse replaced to T (see direct thymine #95)

An example of a non-readable chromatogram


Figure 8."Bad" result example.

It is impossible to interpret chromatogram above properly: more than one sequence is presented on the same picture or DNA sample was dirty: too many peaks.

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© Sophia Veselova, 2017.