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Dear SEQanswers community,
i would like to ask a more general question about exome sequencing, and mutation analysis. I have mainly analyzed transcriptomics data, so excuse me for any naive questions on this matter.
In detail, i have for 3 patients both whole exome sequencing data ( Genomic DNA captured using Agilent in-solution enrichment methodology/paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000) from CTCs (circulating tumor cells) and also exome sequencing data from biopsies of the same patients. Moreover, because both biopsies and circulating tumor cells were isolated from the same timepoint of diagnosis-where the tumor has already spread due to its "specific nature", so it is not definately primary tumor in both. I have both FASTQ files and BAM files for each patient.
The main goal idea, is to identify if there are any "common mutational patterns" (ie.SNPs) between circulating tumor cells and biopsies, in the same patients, which would be very vital mainly for the validation of the CTC isolation protocol (as also for the crusial time of diagnosis of the specific cancer, relative biological mechanisms, etc). However, a major issue is that there is there is only 1 reference normal tissue sample for one patient (that probably limits the identification of somatic variants), as also the small number of patients (6 cancer samples in total), as it clearly limits any discrimination of germline and somatic variants-
Thus, in your opinion:
1) Without any normal samples, i could use a "naive" filtering approach-for instance using firstly mutect2 on cancer mode-, and then utilize various databases such as Cosmic, ExAC as also more resourses, to exlude common germline variants and polymorphisms ?
2) Or alternatively, a less biased approach would be in order to perform a somatic mutation detection, using for both biopsies and CTCs something like a "panel of normals"(PoN) like the methodology described here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561496/) ? or an approach, like the ISOWN methodology ?
3) Moreover, one other approach could be to perform a germline analysis ? in order to identify common variants ? but germline analysis in the context of cancer perhaps is not the primary goal...
3) Finally, perhaps the most "safe" approach is to use for the time being only the 1 patient that has the normal matched sample, perform for this the somatic variant calling in the comparisons CTC vs normal and biopsy vs normal, and wait for any extra normal samples to receive ?
Any opinions or suggestions on this matter would be essential !!
i would like to ask a more general question about exome sequencing, and mutation analysis. I have mainly analyzed transcriptomics data, so excuse me for any naive questions on this matter.
In detail, i have for 3 patients both whole exome sequencing data ( Genomic DNA captured using Agilent in-solution enrichment methodology/paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000) from CTCs (circulating tumor cells) and also exome sequencing data from biopsies of the same patients. Moreover, because both biopsies and circulating tumor cells were isolated from the same timepoint of diagnosis-where the tumor has already spread due to its "specific nature", so it is not definately primary tumor in both. I have both FASTQ files and BAM files for each patient.
The main goal idea, is to identify if there are any "common mutational patterns" (ie.SNPs) between circulating tumor cells and biopsies, in the same patients, which would be very vital mainly for the validation of the CTC isolation protocol (as also for the crusial time of diagnosis of the specific cancer, relative biological mechanisms, etc). However, a major issue is that there is there is only 1 reference normal tissue sample for one patient (that probably limits the identification of somatic variants), as also the small number of patients (6 cancer samples in total), as it clearly limits any discrimination of germline and somatic variants-
Thus, in your opinion:
1) Without any normal samples, i could use a "naive" filtering approach-for instance using firstly mutect2 on cancer mode-, and then utilize various databases such as Cosmic, ExAC as also more resourses, to exlude common germline variants and polymorphisms ?
2) Or alternatively, a less biased approach would be in order to perform a somatic mutation detection, using for both biopsies and CTCs something like a "panel of normals"(PoN) like the methodology described here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561496/) ? or an approach, like the ISOWN methodology ?
3) Moreover, one other approach could be to perform a germline analysis ? in order to identify common variants ? but germline analysis in the context of cancer perhaps is not the primary goal...
3) Finally, perhaps the most "safe" approach is to use for the time being only the 1 patient that has the normal matched sample, perform for this the somatic variant calling in the comparisons CTC vs normal and biopsy vs normal, and wait for any extra normal samples to receive ?
Any opinions or suggestions on this matter would be essential !!