Entering edit mode
2.1 years ago
Saran
▴
50
Hello,
I have RNA-Seq FastQ files of one particular gene from 6 samples. The mice had been CRISPR edited to knock-in this gene. I have been provided a reference sequence of the gene. I would like to know the percentage of mutations of this gene in my samples and analyze the errors. It seems similar to analyses that I have done in the past such as 16S sequencing analysis yet I have never dealt with such a small scale issue of looking at one tiny gene and analyzing variants. How should I go about tackling this analysis? Any programs that would be best?
Thank You, Sara
Can you explain how this was achieved? Are the data truly from a single gene?
Actually, No, I think its just a region of interest from a particular gene. It is similar to analyzing the 16S rRNA gene or ITS region in fungi which I have already done. However, now I will be aligning these sequences to another short reference that I have been given for the region of interest in my reads. With 16S and ITS they were aligned and compared to each other and all of the variants were recorded yet, for this project, I want to compare all reads to the reference. I have paired end Illumina Miseq reads for the 6 samples and together the paired reads cover the region of interest.
If the data are from total RNA or mRNA from entire genome then it would be inappropriate to align just to the gene of interest. One can do it but it is possible that any aligner you use will try its best to align reads there even though they may not have originated from that gene. It would be appropriate to align the data to the entire genome and then look at the gene of interest for further analysis.