Hello, I have some sequencing results I want to blast in order to identify what are known SNPs and what are not and which variations lead to which effect (synonymous or non-synonymous mutation) ? At the end, I want a file with a single Refseq transcript ID (preferably the longest transcript) and all the variations identified for this gene with, for each SNP the indication of the position (coding / non-coding), the consequence (synonymous/non-synonymous) the amino acid substitution etc.. In order to do this I used the SIFT tool on my results. But this latter, seems to choose randomly the transcript sequence he's referring to. For example, when I enter this variations chr19 39191323 + C/T chr19 39191733 + C/T chr19 39195653 + C/T chr19 39196688 + C/T chr19 39196736 + G/A chr19 39196745 + C/T chr19 39207742 + G/A chr19 39214633 + C/T chr19 39215172 + T/C chr19 39215193 + G/A chr19 39218649 + G/A chr19 39219780 + T/C chr19 39220016 + G/A chr19 39220279 + A/T the SIFT tool identifies 13 SNPs in the transcript ENST00000252699 (9 novel and 4 already known) and 1 in the ENST00000445727 but this referring to the same gene : ACTN4! I don't understand why is this happening and how can I force SIFT tool to "blast" the SNP on only one transcript (preferably the one corresponding to the longest isoform) ? I have a second problem related to the same field. This I've got multiple output Ensembl transcript ID for the same gene with the SIFT tool, I tried the AAchange one.Once I had the results, I compared them with the one I had with the SIFT tool. Surprisingly, I found a systematic decay in the mutated codon between this 2 tools ! I check 1 or 2 identified SNPs found with the SIFT tool to see what was the good results and according to the DbSNP database it appears that the SIFT tool is right even if he doesn't always consider the refseq transcript. So my question is why do they identy different affected codon (even when they used the same transcript) and how I can force the AAchange tool to start the analysis at the good nucleotide (to get rid of the decay) ? If it can help you I enclosed the link allowing you to see what I did and what I get :http://main.g2.bx.psu.edu/u/charlotte/h/sift-vs-aa-change <http://main.g2.bx.psu.edu/history/sharing?id=dbac2dd575f62542#> If you could help me on this issues it would be great for me ( my work and my boss)! thanks Charlotte *Charlotte Gueydan *, PhD. INSERM/Université Pierre et Marie Curie Hôpital Tenon - bâtiment de recherche 4, rue de la Chine 75020 Paris cedex 20 tel : 01 56 01 83 75
