DNA Sequencing.  Fluorescence-based DNA sequencing will be provided 
by the Genome Sciences - Genomic Resource Center (GS-GRC).  They 
routinely resequence samples from investigator-supplied samples 
in candidate genes or specified DNA regions.  These are processed
 in sets of 96 (94 + 2 controls) (The minimum sample set size is 
48 (47 + 1 control)).  For this project, (number) samples will be
 submitted to GS-GSC.  We provide information on custom 
resequencing, by providing the genome coordinates to define the 
targets to be sequenced.  The only regions that will be excluded 
are those refractory to PCR amplification or sequencing (e.g., 
low-complexity repeats).  (Include information about your defined
 genes or regions here) Once the region has been defined the GS-GCR 
team amplifies the specified gene(s) or region(s) requested by PCR.
 The PCR primers are designed to specifically target the gene(s) or
 region(s) requested. They are Tm matched and designed from a masked
 reference sequence to exclude design in problematic regions such as
 repeat motifs and high GC content. Each primer pair is "tailed" 
with a universal M13 forward and reverse sequencing primer to enable
 subsequent sequencing. Optimal PCR conditions are determined 
empirically or computationally and confirmed by multiple quality 
assurance steps. Once the regions are amplified, each PCR product is
 sequenced from the forward and reverse direction to provide 
double-stranded coverage. Sequencing is carried out with Sanger Big-Dye
 Terminator sequencing, and detection with capillary-based sequencing
 machines (ABI 3730XL).  Difficult sequences/regions or failed PCR 
are handled as follows: 1) Attempting alternative PCR chemistries and
 thermocycling parameters for the failed region; and 2) if a region 
fails two attempts with alternative conditions, that region is 
considered refractory to analysis. In general, less than 10 to 15 
percent of targeted regions are refractory in this manner. To identify
 sequence variants, the sequencing traces are base-called and assembled
 on the reference sequence, and then a computational tool 
(i.e., PolyPhred) is applied to resolve mixed (heterozygous) 
sequences in the trace (the signature of a SNP/variant). The 
SNPs/variants are identified by confirmation on both the forward and
 reverse strands and, if required, with manual review by specialized
 data analysts.  The only DNA variations that we report are SNPs or 
small (less than 20 bp) insertion/deletion polymorphisms.  The GS-GRC 
produces sequence data and variant calls of the extremely high quality,
 and it well-documented error rate less than 0.5%.  The center will 
provide a defined set of output data that includes the sequence files 
in FASTA format for the regions sequenced, the genotype and frequency 
of each allele for each SNP/variant and insertion-deletion polymorphism
 detected according to position mapping in the human genome, mapping of
 these SNPs/variants in the context of gene structure, and if requested,
 input files for programs like PHASEII, Haploview and PLINK. These data 
files are provided via a password-protected project web site.  They also
 include the data analysis and quality assurance steps necessary to 
identify variants directly from the sequence traces, alleviating the 
need for the applicant to perform these tasks. However, if requested, 
we do provide copies of the Consed-compatible trace files at the 
completion of the study through FTP access or disk copy.