We are proposing to use Agilent SureSelect for whole exome sequencing of four cases with mutations identified in a single gene having 32 exons. Supposing that the SureSelect covers 30 of 32 exons, and they are 75% on target with 90% alignment, all four samples could be done at 100x coverage in a single Hi-Seq lane. The reason for doing so is to show that we can use a NGS method to identify variants normally covered by Sanger sequencing kids in CLIA labs at 10x the cost. If we can, we plan to extend this to an 80-gene panel.
In your considered opinions, is coverage with these parameters sufficient? Further, if we hope to show we can identify 2 or 3 of the 4 mutations in this gene using NGS, are there shared controls available which would be appropriate and available if we wanted to use this sequencing data? Or do you think batch differences between institutions/machines/DNA sample prep differences would instead warrant only using controls sequenced with the same Agilent kit and similar coverage on the same machines at our institution? Better to have solid experiment design before proceeding than have grant reviewers shred us for comparing apples to oranges.
Please let me know your experiences or any relevant publications or edit the above tags. And thanks.