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| Base Calling From a Chromatogram |
Base Calling the report you received from the sequencing lab:
This process will create a FASTA file from the
chromatogram you received from the sequencing lab. Essentially
what is being done is that the program is reading your chromatogram and
turning each peak into a distinct base, G,A,C or T. This is what
is returned to you in the FASTA file, along with a unique ID for each
sequence. If you already have a FASTA file, proceed directly to
the Alignment step. If, however,
you have a chromatogram, you will first need to decide between using
one of two different software options for creating a FASTA file.
There are many options available, but the two recommended choices
are either FinchTV or Phred/Phrap. It is recommended that the
beginner use FinchTV to call the bases for her report. You will
only want to use Phred/Phrap if one of the following applies:
A) you are dealing with a very large report (>50
clones)
B) your report includes multiple reads of a single
sequence, in which case they will need to assembled into a single file.
Note: FinchTV allows the user to see their
chromatogram along with the nucleotide base assigned to each peak but
does not give a confidence value for each base. Phred allows the
user to do high throughput analysis but in doing so doesn't allow the
user to see each chromatogram. Phrap assembles multiple reads of the same sequence into a single sequence.
You can download these software programs for free at FinchTV or at
Phred/Phrap. Either program will lead you clearly through the necessary
steps to call the bases to end up with a FASTA file.
Important Update Regarding Fitch TV:
1. FinchTV is not a base caller. It can only show base calls that are either added by a DNA sequencing instrument or by base calling programs like phred (scf files).
2. FinchTV can show quality values. If a labs has an ABI 3730 and uses KB basecaller, then the .ab1 files will contain quality values and these will be displayed in FinchTV.
Alternatively, if a lab uses phred for basecalling and makes scf files with quality values, FinchTV will display those quality values.
I have some blog posts here that show screen shots of how this works and how phred compares with the KB basecaller at:
Post 1 and
Post 2
Align Your Sequences
Lab Outline
Tutorial Main
Greengenes Main
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