The reference genome in NCBI will not have these mutations/variants/alleles. You will be able to find the referred to original nucleotide sequence. Were did you get the information about the mutations? Also, the cDNA will not have the intron variant, only the gDNA contains introns.

I second Katie A S Burnette on this. If you are new to the field, try to explore the Human Gene Mutation database or Xenabrowser for specific cancers, but none of them can cover all. It really helps to know the source of your mutation list and what your analysis is intended for.

Please refer to attached plot for details of these two mutations, with noting that the gene is on reverse strand and mutation notation is for cDNA sequence. Hope this helps, thanks!

Thank you all for the answers. I am mainly working on cDNA expression and do protein work so yes i haven't got a huge experience on this.

Katie and Anukana, i realize that what i get on the NCBI server are the wild type sequences and i have to get it from there. It is just that i looked for them in the rs sequence server and cannot locate them.

Yong-Dong thank you for your answer. Can you let me know of the method that you used? Did you do some sort of alignment or did you perform an alternative analysis? I would very much appreciate that.

Best wishes,

Nikos

Yong-Dong Wang

Also which tool(s) have you used to get your results (and the plot) that you sent me?

Thank you again.

Nikos Gavalas

The displayed is the reference sequence (hg38) using IGV tool (https://software.broadinstitute.org/software/igv/).

Dear Yong-Dong,

thank you ever so much, i will download the software and have a look. Did you align the cDNA sequence (the cds) on hg38 and got the result?

Best wishes,

Nikos

Nikos Gavalas

IGV is an excellent viewer tool, and for your purpose, no alignment is needed, which also means you need to interpret each mutation for exact location according to the reference sequence given. For instance, the c.192+5 indicates the third codon of p.64 (64 aa, making sure the proper isoform NM_001802, both can be easily located as displayed in the viewer) and 5 bases downstream into the intron region.

Ok Yong-Dong,

i will download the software and have a look. I am not exactly sure what you mean by locating the mutation, for example i would think that i would have to find the cd where it contains the c.192 and then locate the 5th nucleotide on the adjacent intron, but i will look at your approach and attempt to locate them.

Otherwise i will get back to you as you have really helped me a lot so far.

Many thanks for your advice and support.

Best wishes,

Nikos

You can use a graphical interface like mentioned before (IGV) and load a genome, and your VCF files as well as bigwig or bam files.

Thank you Adria-Jaume,

i will look into it over the next few days and i will get back to both of you and Yong-Dong with my progress.

Best wishes for the New Year!

Nikos