I wish to use next-gen sequencing for rare variants in about 800 disease and 200 control samples. The limitation is that total template available for the project is 500 ng total per individual sample. Targeted regions are promoter regions (2 kb upstream) plus short exons 100-300 bp long with adjacent exon-intron boundaries plus 3' UTR 500 bp, totaling maybe 30 kb, distributed over 400+ kb. The exons are pretty far apart for long-range PCR, I think.
Short questions for the gurus:
Does one of the genomic target enrichment methods lend itself to quite limited template like this?
Does the strategy of pooling the samples, then doing individual PCRs for each short fragment, and then sequencing to detect common and rare variants make more sense?
Which template normalization method would work best for shorter fragments, less than 500 bp? For long-range PCR products?
Thank you. George Moxley
Short questions for the gurus:
Does one of the genomic target enrichment methods lend itself to quite limited template like this?
Does the strategy of pooling the samples, then doing individual PCRs for each short fragment, and then sequencing to detect common and rare variants make more sense?
Which template normalization method would work best for shorter fragments, less than 500 bp? For long-range PCR products?
Thank you. George Moxley
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