Resequencing project: What type of sequence to include per gene?

[accipiter]

Hi,

I am embarking on a project that using NGS to sequence a number of human genes (approx 60) and would like some advice around choosing what type of sequences I should be including.

This is for a clinical application and will be carried out on a number of individuals with similar neuromuscular phenotypes who have as yet not got a molecular diagnosis through Sanger sequencing of a smaller subset set of commoner implicated genes.

I'm fairly new to this, and would appreciate some advice around what regions I should be looking at.

Protein encoding sequences: I have selected regions of interest (ROI) based on CCDS genomic coordinates.

Splice Sites: I’ve added 50bp to the 5’ end and 10bp to the 3’ end of each ROI in an attempt to catch splice sites.
Is there any better way of doing this?

Promoters: Not yet considered how to go about this, but I understand there are some promoter databases out there.
Suggestions are welcome

Non-coding exons: This is my current stumbling block. I had originally thought to only include protein-encoding exons, but realize that non-encoding exons exist that may have regulatory functions. Is it common in resequencing projects to routinely include such sequences or not?

..anything re choice of sequences that I have not included?

I’m going to be using the Illumina GAII with 2x75bp paired end long read.

Thanks,

Chris

[frozenlyse]

Are we talking about amplicon sequencing or capture sequencing? For amplicons I would amplify every exon annotated via multiple databases ie refseq + UCSC known genes + ensembl (and maybe aceview if you want pretty unannotated transcripts which have at least some evidence). For capture i would just tile probes along the whole gene +/- 10kb or so