Supplemental Information
Deciphering ADME Genetic Data with an Automated Haplotype Approach
Yingying Guo1*#, Mark W. Farmen2*, Yan Jin3, Hsiu-Yung C. Lee2, Michelle A. Penny4, Kathleen M.
Hillgren1, Stewart L. Fossceco2
1Drug
Disposition, 2Discovery and Development Statistics, 3Clinical Pharmacology, and 4Tailored
Therapeutics, Eli Lilly and Company, Indianapolis, IN
* Drs. Guo and Farmen have made equivalent contributions to this publication.
# Address for correspondence: Yingying Guo, PhD, Drug Disposition of Eli Lilly and Company,
Lilly Corporate Center DC 0714, Indianapolis, IN, 46285, USA
Telephone: (317) 277-4324
Fax: (317) 655-1184 E-mail: guoying@lilly.com
SUPPLEMENTAL INFORMATION
Includes:
Supplemental Materials and Methods (page 2-4)
Supplemental Tables S1 – S12 (page 5-19), S13-15 (page 20-24)
SAS Programs used for Analyses (page 20-91)
Supplemental Materials and Methods
Allele-specific Sequencing
Long-fragment PCR cloning of desired fragments from each individual chromosome was
performed on samples that harbored alleles of interest as determined by the DMET Assay.
Allele-specific sequencing of each individual clone was then performed in order to confirm the
linkage of all the markers of interest. The experimental procedures are detailed below.
Long-range PCR was conducted in a final volume of 50 µL, containing 200-400 ng human
genomic DNA, 400 µM of each deoxynucleotide triphosphate, 200 nM of each primer, 7%
DMSO, 1M of Betaine, and 2.5 U of Takara LA Taq Polymerase (Takara Mirus Bio). Amplification
consisted of an initial denaturation step at 94C for 1 min, followed by 14 amplification cycles
(94C for 30 seconds, 57C for 30 seconds and 68C for 9 min) and an additional 16
amplification cycles (94C for 30 seconds, 57C for 30 seconds and 68C for 9 min + 15
seconds/cycle), and a final incubation at 72C for 10 min. All of the forward and reverse
primers information is included in Supplemental Table 5.
These products were purified and cloned into a TOPO XL PCR cloning vector using the TOPO XL
PCR Cloning kit (Invitrogen) per the manufacturer’s instructions. For each sample, twelve clones
from each plate were picked and cultured in a 96-well format. Diluted cultures were transferred
to a denaturing buffer that was part of the TempliPhi DNA Sequencing Template Amplification
kit (GE HealthCare/Amersham Biosciences). This buffer causes the release of plasmid DNA but
not bacterial DNA. Cultures were heated, cooled, spun, and transferred to fresh plates
containing the TempliPhi enzyme and other components. This mixture was incubated at 30C
for 18 hours to promote amplification of the plasmid templates. These products were then
spun and heated to 65C to destroy the enzyme.
Plasmid templates were then bidirectionally sequenced using the Big Dye, version 3.1
sequencing kit (Applied Biosystems). For each reaction, an appropriate sequencing primer was
used that was designed to anneal to a unique location of the template (data on file). Cycle
sequencing was carried out with an annealing temperature of 50C, an elongation temperature
of 60C, and a denaturation temperature of 96C, for a total of 30 cycles. Sequencing reaction
products were run on an ABI 3730XL DNA sequencer with a 50 cm capillary array using standard
run mode. All bidirectional sequence data were analyzed using Agent, a proprietary sequencing
analysis program (Celera).
DNA Sequencing
A "boost/nest" PCR method was used to prepare templates for the DNA sequencing reactions.
In this method, a boost reaction with a larger fragment was first carried out, and that product
was then used as a template for the nest reaction, using TempliPhi DNA Sequencing Template
Amplification kit (GE HealthCare/Amersham Biosciences) per manufacture’s instructions. All of
the forward and reverse primers information is listed in Supplemental Table 12. Amplification
consisted of an initial denaturation step at 94 degree for 4 min, followed by 26 amplification
cycles (94C for 20 seconds, 55C for 25 seconds and 72C for 1 min), and a final incubation at
72C for 7 min. The nest product was bidirectionally sequenced using Sanger sequencing. All
bidirectional sequence data were analyzed using the proprietary sequencing analysis program
Agent (Celera).