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Throughout the course we have focused on aligning a single sample of reads against a single reference genome with a few exceptions, but sometimes we know that is not the biological case. Sometimes we know that there are multiple different things present in a single sample. Most common situation would be a sample with a chromosome as well as a plasmid. Here we will examine the same sample used in the novel DNA identification tutorial to see how inclusion of the 2nd reference file changes the mapping results.
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The discussion of concepts in this tutorial are identical to the the advanced mapping with bowtie2 tutorial tutorial and work with the same data. The commands in this tutorial will take ~15 minutes. Discussion of results is more thorough in the advanced mapping tutorial. As that tutorial directly assess the difference between multiple mapping types. |
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Why is mapping against multiple references at the same time preferred to mapping against multiple different references 1 at a time, or only providing the reference file containing the sequence where you want to find the answer? The answer relates to identifying real mutations from errors. As we discussed in our initial mapping tutorial/presentation, mapping scores and alignment scores are both related to how confident the mapping program is that an individual read is mapped to the correct location in the genome, and how well that that read aligns at that location.Imagine a hypothetical situation where in you have a 200bp region of a low copy plasmid that differs from the chromosome by a single base.
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- Does it effect the mutations that are called?
- Does it effect the percent of reads mapped as was observed in the advanced mapping tutorial?