If one gene is over 700 bp long then how to get the consensus sequence data from the raw sequence data? I have done the sequencing using both forward and reverse primers.
Also, I want to know the "abcd" of the alignment of the sequences to get the good picture of my data-set. Kindly help me as I am new to this field and I really want to learn.
Frist make reverse complent of sequece obtained with reverse primer and then use Bio Edit software and align the sequences. You can Try DNA baser a free software for getting contings. Use FitchTV to identify Phred Score for sequence aligment.
You need to assemble your sequences, preferably using the reference sequence. Better if somebody experienced could show you once. If you need help you can send me your data and I have a look.
For the purpose, you need an assembler. For de novo assembly, you can use SOAP de novo or MIRA. They are good assemblers. Also, if you want to try on your own, and you have the reference sequence, try local alignment tool, BLAST, and process the result, either by code or manually.
As suggested by the other responses, you need an assembly program. These come with the sequencer you are using, so check with your lab. The assembly program joins two or more raw sequence chromatograms and suggests a consensus, which then can be inspected directly in the program for inconsistencies. After that, you can save the consensus. Unfortunately, while almost all phylogenetic software is free, most assembly programs are proprietary and linked to the sequencing machine. You can of course use any of these programs for any raw sequences (with some exceptions), but if you don't have them available through your institution or lab, they tend to be very expensive. Some programs also allow to store various kinds of sequence data in the same database, so that you can for example go back from an alignment directly to a raw, unassembled sequence to check for problems.
@Arthur: I have not yet seen a good, really free assembly program on the market. The few free ones that are out there (e.g. Staden) do not seem to be as user friendly as commercial ones available, especially if one has little experience or is uncomfortable to work with command line input. DNA Baser is not free. So the situation is very different compared to other phylogenetic software, in that commercial assembly programs are (far) superior to free ones which is why people actually purchase them (often together with the sequencing machine). This is a very similar situation with statistical analysis software, where you can get almost any tool package for free (e.g. in R), but in terms of user friendliness, commercial packages such as STATISTICA or PC-ORD are far superior and therefore do have a good market.
For almost all other types of phylogenetic analyses, there is indeed free software available. PAUP and MACCLADE were few exceptions but they are now replaced by free packages such as MEGA and MESQUITE. For most applications there are R packages, then there is the very powerful and flexible suit of RAxML and related software, plus BEAST and its associates. Plus the excellent alignment programs available such as MAFFT and MUSCLE. And new packages come out almost on a monthly basis. So I think the market is too dynamic for a commercial phylogenetic package to have any chance of succeeding, unless it has a very competitive price (less than $30) and has clear advantages over free software.
I did indeed have problems assembling contigs from various combinations of sequence files and assemblers. In several instances, the assembly program (Lasergene DNA Star) would not display the sequence chromatograms from particular sequencing machines. I am not sure whether this is a problem of proprietary file structure, though.
Hi Biswajit,
Its easy, for alignment, here is Codon code aligner, its paid but comes with trial for 30 days (here is download link: www.codoncode.com/aligner/download/Aligner371_Installer.exe). You can use with full functionality for 30 days, I will guide through the process, here is the procedure after installation.
1. Download reference sequence form GenBank, 2. Go to edit preferences, follow settings as in attachment file. 3. After applying the settings, import reference as well as your sample sequences, those will be in unassembled samples folder 4. Go to Contigs-->Advance assembly-->Assemble in groups (here adjust the parameters as per your lables to sequences) (Note: if you are not able to define names here consensus will not be created)--> click clone (here reverse and forward are aligned together to form a consensus sequence)--> click on the assembled consensus sequence--> File--> Export--> Consensus sequence (It will be exported in FASTA format)
Any issue that comes in between will be resolved, keep posting.
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