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I need to perform tuberculosis mutation analysis. First step I need to do is to align sequences in fasta format against one reference file. i tried CLC genomics workstation --- it hangs on 60 000 lines of fasta reference file for entire day. Please advise what technology and tutorial to use to do that task.

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  • $\begingroup$ I am a poorly trained molecular biologist, so when I build plasmids and have them sequenced I line up the sequences by hand in microsoft notepad. I know it's not a good way to do it, but haven't looked for anything better. What would you suggest for aligning up to a couple dozen relatively short sequences (~500 bases)? $\endgroup$ – user137 Jan 17 '15 at 20:15
  • $\begingroup$ Web VISTA tool designed for short sequences $\endgroup$ – VassiaAlk Jan 18 '15 at 16:51
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I think you incorrectly identified what type of analysis you want. You mention that you have numerous sequences in a fasta file (you mention at least 60,000) that need to be aligned to a single genome. So your not doing a pairwise alignment, your aligning DNA sequences to a reference genome. The fastest known algorithm for this is burrow-wheeler transformation mappings which can be accomplished with tools such as bowtie, bowtie2, BWA, etc...

I'm assuming your experimental design is something like this: You have sequencing data from a number of tissue samples with half or some number of samples being a disease state, the other being normal. So you want to create alignment files for each sample, and than determine where these alignments differ.

After alignment, you will need to create a variant file (VCF) which can be accomplished using samtools or GATK. The VCF file will give you all bp locations with alternative alleles from your reference genome. Which seems to be what your looking for.

Citations that could be useful:

Langmead B, Salzberg S. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012, 9:357-359.

Bioinformatics. 2011 Nov 1;27(21):2987-93. doi: 10.1093/bioinformatics/btr509

Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25:1754-60. [PMID: 19451168]

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25, 2078-9. [PMID: 19505943]

https://www.broadinstitute.org/gatk/about/cited-by

The last url contains 145 publications that used GATK to look at population-scale variant differences. These publications could be very useful to see how other papers have accomplished variant discovery for the use for scientific inference.

For User137 - since I can't comment on this board yet; you want to use a program called muscle. There is an MS-DOS version and it can align short sequences extremely fast.

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  • $\begingroup$ Thank you, I `ll read the recommended literature $\endgroup$ – VassiaAlk Jan 18 '15 at 16:40
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Found a tool: MUMmer is a system for rapidly aligning entire genomes. The current version (release 3.0) can find all 20 base pair maximal exact matches between two bacterial genomes of ~5 million base pairs each in 20 seconds, using 90 MB of memory, on a typical 1.8 GHz Linux desktop computer.

If you know a better programmatic approach, let me know, please

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