Aligning fasta to fastq
I want to align fasta sequences for 28 exons of SCN5A gene with fastq sequence of whole exome sequence from 1000 genomes project.
By aligning these two I want to find mutations within the exons of SCN5A gene.
Should I use conventional NGS alignment methods for this purpose i.e make indices of fasta and then align fasta with fastq or there is any other strategy for this purpose?
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Irrespective of the method you settle on keep in mind that when you align data to an abbreviated version of a genome (in this case just SCN5A gene) there is a chance that aligners will align reads that may not have originally belonged in that region. If that is a cause for concern then aligning to the whole genome/exome followed by retrieving regions of alignment of interest may be a safer option.
and in your case, if any short-read comes from SCN10A which is homologous to SCN5A, it might produces a false positive when only mapped to SCN5A...
Ok I will try to use this but lets say SCN5A is on chromosome 3 then how to get exons only for chromosome 3 from this whole exome sequence ?
You could align to the whole genome/exome and then extract regions you need later: Extracting reads from multiple regions
After alignment I got BAM file. What should be the next step to get the sequence of chromosome # where my desired gene is present? Should I extract regions from BAM file based on chromosome using samtools view and then convert that into fastq? Or Should I convert the BAM file to BED file and then extract required regions ?
Things may be a bit tricky depending on what you want.
Are you looking to get a consensus? Take a look at this. If you only want to restrict to a chromosome or only the SCN5A gene then you could limit the region that you feed to
samtools mpileupby usingsamtools view sorted.bam chr3:1-450000(use the interval you want)