I'm trying to find a program that will do batch sequence alignment.
Basically what I'm doing is, I have a reference sequence obtained from NCBI and I have sequencing files (.ab1) from DNA samples that I've sequenced. I'm wanting to align the sample DNA sequences to the reference sequence to see how well the samples match to the reference.
i.e Say I had a reference sequence for Exon 1 of Gene X (+ and - 200bp) and that I have amplified that exon from 6 genomic DNA samples by PCR, which, after sequencing, leaves me with 12 .ab1 files (forward and reverse for each sample). I essentially want to be able to align the sequences from the .ab1 files to the reference sequence such that I get an output file that has the sequences aligned in the order I want.
So far the best thing I've found seems to be ClustalX which allows me to align multiple sequences, although I have to manually extract the sequences from the .ab1 files, Rev-Com the reverse sequences in "A plasmid Editor", paste each sequence in FASTA format to the same text document and then open that text document in ClustalX. I would LOVE not to have to do all of that for each set of sequences considering I'll likely have to align well over 40 samples with 13 "exons" each - and that's just one population....
The main problem with ClustalX is that when I align the sequences, it throws the order of my sequences out. Instead of being in logical order:
Genome 1 Exon 2 F
Genome 1 Exon 2 R
Genome 2 Exon 2 F
... etc
They end up scattered such that it's a pain to try and get any logical information from the data. -.-
There is a software called dna dynamo in which u can add reference sequence and match them with .ab1/ .seq files. it will align and show how much sequencing happened and does it have any mutation. on tht it will build a reference sequence, which u can copy and blast it to see homology u can contact me if u need more info. i am a regular user of this software.
I agree with Artur that you should clean/assemble your sequences first and then analyse them for similarity to the reference. We do a similar analysis when we engineer our HCV clones: full-length sequencing with several sequence files (up to 40 in some cases). Our tool of choice is Sequencer, it allows you to clean up the ab1 files, assemble them using a reference sequence, revise the consensus sequence, and export it in the form you prefer, all in one package. You could even perform alignments within the program itself, once you have the consensus sequence(s).
Many other software can do the same, and I would bet your department has access to some of the commercial ones (typically Sequencer or Lasergene). Good luck.
Phred, Phrap and Consed are some of the most established tools. Phred for base calling, Phrap for assembly, Consed for viewing and editing the assembly. Phred will assign a quality score for each base called, but it's often worth viewing the sequencing trace for low quality calls with something like 4Peaks
Ok! Thanks for the responses everyone!
So I'm still fairly new to sequence alignment (being in my honours year), but from what I understand from your responses:
- I need to use a program to assign bases to the chromatogram peaks. So something like bioedit?
- Then use another program to merge the forward and reverse sequence reads for each sample - creating a contig.
- Then trim the poor quality regions (usually the ends).
- Then align the sequences.
I've found a program called GeneStudio that's free and seems to do all of these things . Has anyone used it/can give feedback?
Hi
you can use software called ATGC which align you all files and build up one sequence and same time you can see the peak of each one and edit if there are any error in sequence. It has many options.
SeqMan Pro from the DNASTAR Lasergene package is a very good assembler. You can control the sequence name order and provide any reference you want to serve as a backbone. Works directly with chromatograms.
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