I have sequenced a 16S rDNA sequence and got one ab1 file of chromatogram. Please tell me which software or online tool I can use to check the chromatogram?
Hi Kunal,
As mentioned above Bioedit is a good one. You canl also use Chromas or DNAwin for viewing and editing chromatograms
bye
There are a lot of different software for analysis of sequences. BioEdit, Chromas PAUP, SeqAssem, Staden and many others are suitable for doing this.
Hi Kunal,
you could use ApE (A plasmid Editor): http://biologylabs.utah.edu/jorgensen/wayned/ape/
Open AB1 file with this program. Go to "File" and select "New DNA from Basecalls" or press "ctrl+shift+N".
You'll get the sequence as text, which you can blast or compare otherwise.
The program is also good for cloning analysis, finding reading frame etc.
And it's free!!!
Best,
Kai
Dr. Kai,
I want to check the purity of the sequence. I have both sequence file and ab1 file. with ab1 file I have constructed the chromatogram in BioEdit software. Someone has told me to check the purity of sequence through chromatogram.
Would you please tell me how to read that chromatogram. I have got different colored peaks for each similar type base pair. How could I know whether the chromatogram is correct or not? What means double peak or unclear peaks and how to check it? Also I am attaching the photograph of part of chromatogram for kind concern. Thanks.
Hi Kunal,
since the sequencing reaction (dideoxynucleotide method by sanger) is done with polymerases which do not have proof-reading activity, but high fidelity, it is normal to have background peaks. To check if the sequence is correct, blasting seems to be the best method.
I guess you want to check, if the sequence is correct when talking about "purity". But you have to keep in mind, that procaryotic genomes contain between 5 to 15 copies of genes coding for 16S RNA. If you have mutations in some of these, this might result in double peaks since these bases are polymorphic within a single cell.
If you are using a sequence which was subcloned into a vector you might want to analyse other clones or repeat the sequencing reaction.
Best,
Kai
As I can see on the fragment of your chromatogram, you have presence of multiple signal. This may be caused by different factors - contamination, presence of multiple copies of analysed fragment (16S usually have some copies with few diffences between each other, also pseudogenes may be occured), degraded primers and so on. But you can analyse it by extracting main signal and checking its correctness by BLAST. If you want to obtain clear sequence without secondary signal you may repeat PCR and sequencing. If you have possibility to clone this fragment, this can be helpful too. I attached to you the exampe of chromatogram without any secondary signal.
Best wishes
As Abrar suggested, Chromas Liteis easy to use and free of charge, so it is good to start with. When your need for sequence data processing goes further, Geneious 7.0 provides diverse tool for chromatogram editing, alignment, assembly, creating phylogeentic trees, BLAST seraching, submitting your sequences to databases etc.
There is 14 day free trial, full program is quite expensive though.
Dna baser software is their for sequence editing. It will easy to handle. U can download freely from google search and handling manuel also available. Compare with other software, it s more easy to edit. Just try it.
Since you performed sequence run on AB platform, it is best to use Seqscape software if you are interested in identifying SNP other wise use bioedit software go for sequence assembly and perform BLAST using NCBI server
I use Geneious Pro (http://www.geneious.com/). It's by far the most intuitive piece of software I've use so far. As Jouni Heikkinen notes, it is pretty pricey. I was recently very impressed by the CodonCode aligners toolkit (http://www.codoncode.com/aligner/) as you can assemble contigs and has better tractability than Geneious. Both have free-trials.
It's always good practice to check chromatogram of sequencing results, as some time miss base-calling do occur. Chromas is very convenient. For nuclear loci, you can make a primary idea about heterogeneous sites. For your 16S rDNA , I guess you might use in phylogenetic purpose. In such case, you can just code the double(multiple)-peak base as ambiguous site, i.e. S, W, N.
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