Hi

I would suggest codoncode aligner.

For one month you can use demo version. It is perfect. Just go here and enjoy it:

http://www.codoncode.com/aligner/

all the best,

Hi,

I've used several versions of Sequencher, which works fantastic, but unfortunately not free (and not cheap) and Bioedit also works pretty well.

I would suggest Chromaseq which is a package in Mesquite that allow you to process chromatograms, edit your DNA fragments, makes contigs... Here it is the link if you want to check it out

http://mesquiteproject.org/packages/chromaseq/manual/index.html

I use Bioedit, it's free and very easy to use!!!

If you are talking about genome assembly by this Next Generation Sequencing (forward and reversed reads), then go for Velvet and SOAPdenovo softwares.

For a smaller set, I use CAP3 at the following webpage (http://pbil.univ-lyon1.fr/cap3.php) . It will show an alignment with each of the sequences that were included so you can see how well the sequences were put together.

To generate consensus sequence using the fasta sequences could be performed by many software, but using .ab or any chromatogram files would be better performed by commercial softwares. At par, staden is performing good in trimming, building consensus, comparison of chromatogram in same window etc. (guide: http://www.bioinformatics.babraham.ac.uk/training/Staden%20course.pdf) (thanks to my colleague Charles Lamien)

I use Bioedit, it's free and also SeqScape but not free

It sounds like you already have the sequences of a PCR product, sequenced from the forward and reverse primers, in BioEdit. If this is true, you can easily convert the reverse sequence(s) to forward by selecting the reverse sequence(s) and then using the pull-down menu for Sequence:Nuleic Acid:Reverse Complement

This will "invert" your reverse sequence(s) so it (they) should now align with the forward sequence(s). You can use CLUSTAL which is built in to BioEdit for this, under the Accessory Application pull-down menu.

Once all of the sequences are aligned, you can easily highlight sites where not all of the sequences are identical using the pulldown menu for Alignment:Plot Identities to first sequence with a dot. Then you can decide which sites need to be checked in your original chromatograms to decide whether or not to edit a sequence.

BioEdit will also produce a consensus sequence with the pull-down menu item Alignment:Create Consensus Sequence, but it may be better to edit incorrect base calls first.

You must remember if your sequences come from sequencing must see the edges usually have noise, after that do a CAP contig of the sequences and create the concensus sequence of the result I use BioEdit for this kind of analyses is simple and fast. Best results

BioEdit is a good tool for small sets of data. But if you don't want to download the program and install it in your computer, you can use CAP3 (that is the algorithm that BioEdit use to generate the consensus sequence from a forward and reverse sequences). If you have quite a lot of data (some times I have sequencing results from several hundred clones at the same time) I recommend you to use CodonCode (the free trial has the option already).

You can download a free trial version of codon cuode aligner sich paste for one month per user. This is easy to use and you can export your sequence in the desired format.

if you have a reference and want to sort your contig I suggest using MAUVE. http://asap.ahabs.wisc.edu/mauve/

Why this software will automatically generate reverse and forward output.

What is the size your sequence..? If it is less than 3 Kb we can align through DNA star (Laser gene - Seq man). It is easy to use.. of course you can use mega 5 also.

Thanks all for very interesting suggestions. My desired sequence size is 1100+ bp. cyt-b. Not genome. The forward and reverse are around 500-1200.

Then you can align with DNA star itslef. It is easy to do.

I agree with Dhanasekaran. In DNA star Megalign option can be used to get the consensus sequence.

Ashok,

I'm just a beginner in bioinformatics. However, perhaps you could get information on the web on the Unix program named Velvet.

In the case that you have a sequencing project, with several samples to be analyzed, I will recommend you, the STADE PACKAGE, because it is a fully developed set of DNA sequence assembly, with editing and analysis tools. Furthermore, it is available for Unix, Linux, MacOSX and MS Windows, and It is free.

Is one of the best tools for sequencing projects, you can do almost anything.

http://staden.sourceforge.net/

I agree with Marcelo Nazabal...

Try ApE! http://biologylabs.utah.edu/jorgensen/wayned/ape/

Until now I have got good experience with BioEdit. It allow us to see and edit the sequence along with the histogram. It is also easy to copy the desired sequence directly from the histogram file which is very helpful while working with forward and reverse sequence of longer amplicons.

You can keep using bioedit it is pretty good for consensus sequences along with editing of mismatched bases and i got good results from it.

i am using Snapgene for sequence alinement. so you can also use it. although its not free. but trial version may help you a bit.

To assembly contigs I use and recommend geneious software (http://www.geneious.com), it is one of the best and friendly softwares I know. Although it's not free, it is much less expensive than most of others competitors.

I have used SeqMan and SeqMan Pro. these have been very useful and easy to use, but am informed that is no longer free.

Biolign, a free sequence analysis tool might be used for this purpose. It is the updated version of Bioedit.

Does anyone have good experiences on DNA laser star, i forgotten the way to edit sequence with chromatography view. It means two windows are open together: text sequence and chromatography

How good is geneious and what is the percentage quality that is needed for a sequence/contig assembly, so that we can rely on it?

Which is sequencing system used this work?

Because, my experience are next generation sequencing, i.e, 454, ION and Illumina. I usually use MIRA (http://www.chevreux.org/projects_mira.html) and Newbler of owner Roche to assembly and consensus sequence.

I too use, Geneious (http://www.geneious.com) software to edit sequence, but is necessary buy it, is not free or open source.

Hello dears,

I thing I have same question but in different expression. I got FP and RP for a particular gene from sequencer machine (dideoxy method). I seek to join them into one intact sequence. Can you help me in this matter. Please write in detail.  Usually I am using BioEdite program.  

One more question is (Does consensus sequence is contig sequence ?). 

Thank you

Yes, you can try Mega 5.0 and the latest which is MEGA 6.0. Submit all your sequences and click the reverse complement for your reverse sequence so that it will give overlapping aligned sequence with its forward complementary sequence. MEGA 5 or 6 is a free ware. Another method is by submitting your reverse sequence to SMS (Sequence Manipulation Suite) Reverse complement to give you the complementary sequence. Then you can align forward and reverse sequence using pairwise alignment and your  FINAL SEQUENCE is the overlapping sequence. Do this to each DNA sample....Then to each sample you can submit for Multiple Sequence Alignment. You can use MEGA or you can use using T-coffee, Clustal W or MUSCLE.

maybe you should try SeqTrace ?.I'm working on the similar problem too. wish it help. 

The best software would be ChromasPro! Good luck!

I have used SeqMan Pro from DNA star. Although it is not a free software, you may have a three months free full trial. I have used that and other software that come with it.

You can use the Staden Package, available in this link.

http://staden.sourceforge.net/

I can recommand you Geneiouse (trial version is free)...you can make de novo assembly or map Fw and Rev seqs to reference sequence...and it is appropriate as for seqs from Sanger as well as NGS sequences....it is easy, fast and very intuitive :) good luck!

It is my old post! 

Hello everyone,

I have a doubt related to sequencing. I send a few samples (particular gene PCR product) for  sequencing but the sequence that I have got after doing BLAST, its showing 16s rRNA gene similarity with many species. The doubt is I am using particular gene primers to find specific genes in my organism which is already  is present in other bacteria.  After BLAST I should get that particular gene name rather than any 16s rRNA sequence that is the result of universal primers. Please  suggest some scope. Thanks in advance.

Hi Anupama Rani,

There are homologous conserved region exists in every organism which gives you some kind of sequence similarity when you blast. Even if your sequence is not available in GenBank, your sequence is going to match with some available closely related sequence of other species which will show you 100% similarity. Also, 16S has lots of ambiguous sites due to loop present in its structure. Remove the ambiguous sites and blast it.  

Thanks Ashok Sir.

Even I heard of removing some part from that 16s sequence to get the gene sequence. Can you please explain me how to do that. Also some sequence  could not show any similarity search  result in BLAST. Is this the usual case while gene discovering?

Align your sequences in some alignment software (i.e MEGA 6) with available sequences of closely related species (sister species) from GenBank and chop out ambiguous sites. I guess your interested GenBank sequences must have corrected and published as in my case.  

Hi!!I think MEGA v.6 is good. But before it, you need use SeqMan for template DNA sequence that thrives reverse& forward primer alignment properly. Then anyone can use nucleotide blast for specific(16S) for species identification~~Thank you

please  I need to use saqman program

Marivaine! It is very easy using BioEdit!!

1) Save the sequences in a txt file and import them.

2) If necessary, you can select the reverse sequence set the "reverse and complement" command (shift+control+R) if the sequences came from a forward and a reverse primer...

3) Click in: Accessory application > CAP contig assembler program... A window asking for the parameters "length of overlap" and "% of complementarity" will appear (the default are 20 nt and 85%)...

4) Click enter, and enter again (after a DOS window appear)...

After this steps, the contig will appear together with the sequences ina new window... You can select the first and the last nt and copy the entire contig...

also i have the sequence of forward  and the sequence of reverse i want to make combination of both to enter them on NCBI 

DNA baser assembler is good but not free you can still cite it even using the trial version. Codon code aligner can also be used but again it is not free. The best thing would be to find the contig and blast it to find any changes with respect to the reference sequence.

Thanks to you all for your contributions. It really helped me out with similar problem.

Thanks all researcher for your kind suggestions and recommendations.

You can also use DNAdragon for contige construction. I have also used this and this software is very easy to use and make good contiges..

I am pasting the link below, from where you can download it.

http://www.dna-dragon.com/

Hi! You can also use GeneStudio, it is a free software.

I use MEGA 7 for sequencing and alignment.

I also use bioedit for sequence alignment as it was correctly explained by Paulo Ivan Fernandes Júnior .

In Bioedit software accessory application there is cap contid assembly option in which you need to take forward as well as reverse compliment seq it will give matching seq can be use for further analysis.

Hi,

with Bioedit you can do it perfetelty

Hi, Can somebody of you kindly tell me how to make the consensus sequence from forwad and reverse sequences of ND4, where for some sites two bases with identical peak heights in the chromatogram are present? I am using Bioedit.

thanks a lot!

Dhanusha Nirmani, in diploid organisms it is common to have two bases present at one site, from the two alleles. A site that is A in the allele from mom and G in the allele from dad, can be encoded by "R" for purine. The other two base codes are all listed in the IUPAC table in the link below.

https://www.bioinformatics.org/sms/iupac.html

Brian Thomas Foley, Thanks a lot for your fast respone. I agree with what you told, but I am afraid when it comes to mitochondrial sequences, can we apply that? Actually i am trying to use a set of ND4 sequnces to infer on phylogeography of Aedes aegypti mosquito populations. So i need to come up with consensus sequences of forward and reverse sanger sequences of this mosquito mitochondrial region.

Hi,

Bioedit can do it perfectly

Hi @Abdoulaye Soumare, yeah I am using Bioedit. My question is since i cannot have two consensus mitochondrial sequences for one sample, which base I should select from the two different bases at the same site given that the two peaks of each base are of mostly identical height? For example lets say for one sample my forward sequence has a T at 120th positon while the reverse sequence has a A at the same position. When i check the chromatograms, both chromatograms have the two peaks at the exact same position with mostly identical peak heights.

Dhanusha Nirmani it depends on what type of organism you are dealing with, and what you need the data for. Some organisms can have two or more different mitochondrial genomes although I think it would be reare for them to have an exact 50:50 mix. Also many organisms have some nuclear copies of mitochondrial genes (numts) but again the mitochondria should outnumber the nuclear copies and not produce an exact 50:50 mix.

If you need the data to determine the species of you sample, a few R, Y, W or other mixed base IUPAC characters may not hurt. If you are looking at population structure within some geographic area, it may be more important to get every base right.

If you gene in your organism should be "pure", then the mix indicates a problem, most likely PCR contamination. PCR contamination happens to everyone, it does not indicate "sloppy work". The very best labs can nearly eliminate it, but all labs need positive and negative controls constantly run to ensure that if any contamination happens it is caught early before any efforts are wasted analyzing contaminated results. Although every cell in the organism contains a few mitochondria, the PCR pruct produced by PCR ends up many millions of fold more concentrated than any sample DNA (the molar concentration of the product is enormous even if the micrograms of DNA per volume are low). I have heard of proven cases where PCR product from a lab on a different floor of the building contaminated a lab, carried in by a coworkers contaminated glove or something. All labs need to do PCR set-up in a different room from the site where PCR is done or PCR products are handled. Just opening a PCR tube can create aerosols which spread PCR product around.

Brian Thomas Foley Thank you so much for your detailed clarification.SO you are suggesting me to remove those samples from the analysis or do you have any idea of correcting them (with some logical method or any software) and getting the most probable base at those particular sites? As Iam dealing with a small number of samples, it would be great if I can get them corrected and use them for the analysis.

Dhanusha Nirmani I think it depends on how certain you are that there is no PCR contamination, how many of these "mixed sites" there are in the data, and a few other things. I am no expert on mosquitoes, but I am guessing that there is low diversity within any one species within one mitochondrial gene. With humans, for example, we have only a few informative sites in the whole mitochondrial genome, and tracing mitochondrial DNA ancestry in humans is not quite the same as tracing chromosomal DNA markers along with mitochondrial sties.

i have forward and reverse sequence how can I convert them into consensus sequence can anyone tell me

Yalavarthi Nagaraju You can easily do that with Bioedit. First open the forward sequence (ab1 sequence) with bioedit. Next import the reverse sequence (ab1) to the same window (File>import>sequence alingment file). Then reverse complement your reverse sequence (sequence>Nucleic acid>Reverse complement). Select both sequences & align using ClustalW (Accessory Applications>ClustalW Multiple Alignment). you will now see that the two sequences are aligned together. Look for the best aligned region of the alignment. Check the aligned region with the chromatograms of both sequences for any ambiguities, if present you can manually correct them in the alignment. (Remember to look at the reverse complement chromtogram of your reverse sequence). Copy the bases of the reverse sequence from the begining to the middle of the best aligned region. then paste it to the complementary region of the forward sequence. (Lets say you copied from base 1 to base 250 on the reverse sequence. now paste it on the same region (base 1 to base 250) on the forward sequence. you are actually replacing the bases of that region on your fowrd sequence). now you can delete your reverse sequence. in the same way, import your foward and reverse primers to the alignment window (primers should be in a compatible format. ex: fasta). reverse complemnet the reverse primer and manually align it to begining of your consensus sequence and find the edge of the sequence. same way you can align the forward primer to the end of your sequnece to find the end of the sequence.

Dhanusha Nirmani You have explained clearly, I have also the same problem, I donot know how to solve the problem, I have got forward and reverse sequences for a ITS gene, from the company, using forward and reverse primers, I want to draw phylogenetic tree and want to correct these sequences, What will I do? Can I make a consense sequences by combining these two sequences, if yes how? Kindly explain this more,

Aqleem Abbas Yes. You can follow the same steps I have mentioned above to make your consensus sequences for each pair of forward and reverse sequence.

In addition to the above good suggestions, I have been using geneious software for my Sanger sequence analysis (https://www.geneious.com/). With geneious its pretty simple to select and align both your forward and reverse sequences, then select a command to generate consensus sequence, I will suggest you try this software too.

Dhanusha Nirmani Thanks. A researcher say there will be point mutations or errors from the sequencing company, in the forward and reverse sequences, how to remove this, I mean Will I align the sequences or assemble the sequences? and what about peaks? Two peaks over a base means there is an error, is not that? I am very confused. What I want to say, I used the forward and reverse sequence to make consensuse than I want to blast, then want to make a phylogenetic tree,

I have some blog regarding phylogenetic tree construction, this might help in some way... albeit it is for beginners...

here is the link...

https://hubpages.com/education/DNAge-Molecular-Dissection-of-Species-Identity

Madison Pascual Munar Thanks for sharing, I have two files abi files from the company for forward and reverse sequences, I want to remove mismatches and check quality issues. and then want to make a consensus sequence, If you know how to do? Kindly write in detail here or show me screen shots

Aqleem Abbas SnapGene (SnapGene Software, www.snapgene.com) can be used to check chromatogram quality and to guide quality trimming. High quality reads can be assembled using CodonCode Aligner (CodonCode Corporation, www.codoncode.com) and assembled sequences can be used for BLAST analysis (NCBI).

my paper might help here is the link:

Chapter Biomineralization of Metallic Tellurium by Bacteria Isolated...

if you have other specific questions, feel free to message me

Dhanusha Nirmani is right, we can use bio edit to do the analysis.

I would recommend DNA baser (https://www.dnabaser.com/) or CodonCode Aligner (https://www.codoncode.com/aligner/tutorials/index.htm) to edit, trim, view, and assemble your Sanger sequence data.

use bioedit software to generate consensus sequence it is simple to use

Like Yalavarthi Nagaraju has mentioned, make use of Bioedit software which you can conveniently download. It is easy to use. Save your sequences in Notepad(*txt format) and import. Follow the steps as stated by Paulo Ivan Fernandes Júnior above.

It is quite easy to use

Adewale A Oke I just want to ask what if the consensus sequence has some mismatch in it, should I carry on with blasting or first I should edit it?

Zenande Makala .First edit it

edit it with Bioedit

I'd like to bring another program suggestion aside from BioEdit, which I never learned well how to use. Its a very similar free open source program called UGENE (https://www.ugene.net). It's full of interesting features and it easily produces consensus sequences from multiple sequence alignments in a tab to the right edge of the program's window. You can choose if this consensus is gapped or not, if this consensus is strict (prohibiting ambiguous nucleotide symbols e.g. K, Y, R, H, D, M, N, etc.) as well as other patterns built therein. I found it much more intuitive and easy to use than BioEdit. I had the same problem to obtain a msa consensus sequence and I did it with this program very easily, I hope you like it.

You may map it to the reference sequence

You can use bioedit

You may also use PHYDE, a free and very simple tool http://www.phyde.de/

I get the same problem with MEGA: how can you generate the consensus sequence from an alignement ?

Bioedit is very useful for that