Recently, I saw Bernd's work which analyses the difference between one MSI-CRC and MSS-CRC with NGS method. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008745/).
My interest is how to use Whole Exome Pyrosequencing (WEP) to capture the germline mutations, after all they just used WEP to test two tumor samples and their benign samples.
I am sure that they could get total somatic mutations using comparison between the tumor and benign sample, but what about the germline mutations?
Germline mutations require less sequencing coverage than somatic mutations and should be detected in the benign tissue.
Germline mutations will be present also in the non-tumoral sample. Thus, the comparison of the non-tumoral sample BAM file with the reference genome (NCBI) will pick them up, together with the SNPs of that particular individual. In order to ascertain what is a "mutation" and what is a regular SNP, I think the most straightforward way would be to check their population frequencies in the hapmap/1000 genomes project. Subsequent functional significance prediction ( (PolypPhen) may be very useful to further classify the mutations.
Thanks Dina! Whether they identify the germline mutations, like Sergio said. Comparison performed between the sequencing data of benign and the reference data (e.g. NCBI) to find out the candidate mutations. And then drop out the mutations which recorded in NCBI of 1000 genomes project. Finally, the remain mutations are germline mutation. But I still suspect, just two benign sample and two SNP data (NCBI & 1000 genomes projects) is enough to find out the mutation by NGS technique?
Yes, that should suffice. We did NGS and compared to the normal genome to find mutations. Then we compared the list of mutations to dbSNP, 1000 genome , followed by insiico tests such as polyphen and mutationtaster.
Usually, if you consider any germline mutation, it would be ideally of 50% depth frequency of 100% depth frequency. (not valid for mtDNA). So, for capturing germline mutation, one need not have very high depth of coverage. Thus, its easy to validate those mutation with Sanger's sequencing too. Again, it is expected to find similar pattern in normal tissue too.
- Badges
- Science method