We got forward and reverse sequence file for the samples we have send to Macrogen (Korea) for 16S full rRNA sequencing. The sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA). Universal primers 785F (GGATTAGATACCCTGGTA) and 907R (CCGTCAATTCMTTTRAGTTT) were used for sequencing and another set of universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) were used for the amplification.
Now, i want to get accession number for those sequence from NCBI/EMBL/DDBJ. It seems the sequence needs editing as they have provided us two files, forward and reverse sequence result.
Ques 1 : How can i merge forward and reverse sequence data and formulate a single full 16S rRNA sequence, edit/trim it and upload to the database so that i can get accession number for adding in our journal ?
Ques 2 : Also, can anyone please tell me how to do cladistic analysis after editing/trimming those sequences ?
Hi Dr Pandey,
Greetings,
Regarding your query, I wish to inform you that you need to do some bioinformatic analysis before submission to GenBank and to get Accession numbers. First you have to manually examine the sequences individually using BIOEDIT, i.e. to check both the sequences and do editting at places where there is an ambiguous nulceotide, by comparing the electropherograms. then you have to reverse complement the reverse sequence and align with the forward sequence. Then trim the terminal sequences which remain unaligned. this way you can get one sequence for each isolate. then submit the sequence using SEQUIN tool from NCBI. for phylogenetic analysis use MEGA 6 or 7.
best wishes,
regards
SD
Greeting Dr. Datta,
As you have suggested, i have checked electropherograms (.ab1 file) and matched with the sequence file provided (.ab1.seq) and it seems all the peaks are present for the sequence file, that make me conclude that the sequence file (.ab1.seq) provided by Macrogen is pre-edited (Please correct me if i have reached a wrong conclusion). Then, I have used Bioedit (V 7.2.5) and trimmed unaligned terminal sequences. Sir, what shall I do for those unaligned/better mismatch sequences (in some there are few and in some there are many) in BETWEEN/MIDDLE while matching those forward and reverse sequence. Since it is a deNovo sequencing so unaligned sequences are more as Universal Primers are used.
What do you suggest, that i should do in order that i can submit to NCBI and they can easily accept in their review.
With Regards,
Binayak Raj Pandey
https://submit.ncbi.nlm.nih.gov/subs/genbank/SUB4150191/subtype
It is quite simple to use. All you need is, to submit the proper information about the stain, its isolation process and the sequence procedure which is being adapt to get the data, and in case if you want to edit the previously submitted data, I think you can send a updated sequence via mail to NCBI with the old accession number as a reference. You can use the above mentioned link for the submission.
Thank you
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