thanks Mr. jamal. but i read in one paper they have written "DNA sequences were aligned by ClustalX, then subsequenctly optimized by eye (e.g. gap-filling, nucleotide editing)". thats why i am confused. or maybe its call optimize instead of editing! thanks

Thanks. But as i can see from my sequence data, all sample's sequence are not same lenght, for example, some are 410bp, some are 415bp.... So when i put all sequence all together in notepad, then its look different shape..so is that ok and i can perform analysis with that shape of data? Thanks

if you want to made a phylogenetic tree, short differences are not so important (5 bp, as you said). However you can select the extra end of the long sequences (after alignment) and delete them.

thanks Parisa. let say i can delet the extra end. but how about the gap? for example, which started from first of the sequence. i need to run Arleqin software to find the haplotype diversity, AMOVA test etc besides making phylogenetic tree. thanks

These matters are discussed in this publication:

https://www.researchgate.net/publication/235601668_Incorporating_molecular_data_in_fungal_systematics_a_guide_for_aspiringresearchers?ev=prf_pub

Article Incorporating molecular data in fungal systematics: A guide ...

If you want to construct a phylogenetic tree you should modify your alignment results. You can run Gblocks software, which is available as a standalone and server options (http://molevol.cmima.csic.es/castresana/Gblocks_server.html). Gblocks eliminates poorly aligned positions so that it can be used in phylogenetic studies.

thanks all. and thanks to Bodhisattwa dada. it's really nice software.

Yes, you need to examine the quality of your alignments before using them in downstream analyses. Errors may arise due to deletions/insertions in the sequences of your interest. Multiple sequence alignment editors allow you to edit the alignment (not the sequences) to correct such minor errors and obtain the optimal alignment for the phylogenetic analysis or so. You may try Jalview.

http://www.jalview.org/

I suggest use of Geneious software, It's proprietary, but not expensive.

- http://www.geneious.com

thanks all. i use BIOEDIT. but how and which base i should fill/delet..thats i don't understand. let say i have here for 4 sequence as example:

CAAA..................................C...............GGA- -

-AAA....................................T.............GGA- -

AACA....................................T..............GGAAA

- - CA.....................................T..............GGGA-

what should fill or delet this gap (only first 4 base, middle one and last bases). in between means..there is other sequence. thanks

@Dr. Rene, i downloaded the MAFFT but can't instal properly in my windows! can't find the mafft.bat file also which has mentioned in instalation guide. hope to hear from you. thanks

thank you sir @Dr. Rene. i will use online version then. how about the Mesquite, i think they don't have any online version for it. anyway, could you please make comments on my DNA base in top of your answer regarding editing the aligned sequence. thanks

and how i cite for MAFFT, can't see anywhere? thanks

thanks all. i use BIOEDIT. but how and which base i should fill/delet..thats i don't understand. let say i have here for 4 sequence as example:

CAAA..................................C...............GGA- -

-AAA....................................T.............GGA- -

AACA....................................T..............GGAAA

- - CA.....................................T..............GGGA-

what should fill or delet this gap (only first 4 base, middle one and last bases). in between means..there is other sequence. thanks

MAFFT can be used online also. No need to download.

Following links can be used:

http://mafft.cbrc.jp/alignment/server/

http://www.ebi.ac.uk/Tools/msa/mafft/

http://toolkit.tuebingen.mpg.de/mafft/

Citation: Katoh, K., Kuma, K. I., Toh, H., & Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic acids research, 33(2), 511-518.

Don't worry about the gaps as long as your sequences are aligned optimally. If not, just move the nucleotides/gaps of concern to the optimal position according to the 'whole picture' of all sequences aligned.

Thanks Wafa Habbal.

I suggest you to read first one of the basic Bioinformatic books like for example "Bioinformatics for Dummies". It can be downloaded for free from the internet in pdf, just Google it. For sure you will find answers for all your questions.

Please read the user manuals of software. They are easy to follow. See below on BIOEDIT

http://www.mbio.ncsu.edu/bioedit/biodoc.pdf

Try also GeneDoc or Jalview.