**FIND Quality Control Procedures**

**Clinical Quality Control**

## Introduction

The goal of the Clinical Quality Control Committee is to develop
standard procedures for the collection of clinical data. For a
multi-center study, the most important step in quality assurance for the
collection of clinical data consists of the training and certification
process. In FIND, this training and certification will be done at each
PIC.

## Medical Chart Review

> PIC technicians are trained and certified for medical chart
> abstraction by PIC coordinators. Five medical charts from each PIC
> abstractor are xeroxed and sent to the GADCC within the first month of
> the abstractor beginning data collection. These charts are then
> reviewed by data abstractors from other PICs, and Kappa statistics
> calculated for entry criteria by the GADCC to determine the
> reliability of the data. This process is repeated annually for each
> data abstractor.

## Blood and Urine Collection Processing

### Staff Certification Requirements

The blood drawing and preparation are performed by certified
technicians. Each institution or organization is responsible for
ensuring that technicians are trained according to institutional
guidelines.

### Participant Compliance with Protocol

A fundamental function of data collection in FIND is to provide accurate
phenotype assessment for diabetes and renal disease. Hemoglobin A1C and
history will be used to assess diabetes phenotype. Fasting blood glucose
needs to be determined in all participants without ESRD who have an
indeterminate Hemoglobin A1C level, per the FIND protocol. In addition,
it should be obtained whenever possible to confirm the absence of
diabetes. To obtain valid and comparable measurements of fasting blood
glucose, FIND participants should fast overnight for at least 8 hours
before blood is drawn. Interviewers who administer the medical
questionnaire are trained to explain the importance of compliance when
recruiting FIND participants and to obtain their agreement to comply.
When FIND participants are contacted before their appointment about the
scheduled visit, they are to be reminded of the fasting restrictions.
Time of the last meal will be recorded. The GADCC analyzes study data
for information on length of time fasting and reports the percent of
participants at each PIC who do not comply.

### Maintaining Proficiency

To maintain their proficiency, technicians are urged to perform blood
drawing and processing at least once each week (or 8 times each 2
months). The GADCC analyzes the study data to assure that all
technicians collecting and processing blood in the PIC are performing
these procedures frequently enough to maintain their proficiency. For
PICs using vendors, maintenance of proficiency is dependent on the
vendor's protocols. PICs using hospital-based technicians will need to
comply with JCHO (Joint Commission Hospital Organization) requirements.

### Packing Samples for Shipment from PIC to NCI

All vials of blood samples, as well as the plastic bags that contain all
samples for a given participant, are labeled with the participant's
barcode ID. A shipping list for all sets of samples is enclosed with
each shipment to NCI. The person unpacking these samples at NCI verifies
that the IDs on the vials match the ID on the plastic bag and the ID on
the shipping list. If any discrepancies are detected, NCI contacts the
PIC to resolve the problem.

Blood vials shipped to NCI must be packed securely to avoid both
breakage and warming. NCI monitors the arrival condition of the samples
sent from each PIC. If problems are encountered, NCI notifies the PIC
involved.

Shipment of all samples is coordinated through the GADCC website. To
avoid delays in transit to NCI which might cause samples to be warmed or
thawed in shipping, it is especially important that the NCI is made
aware of samples shipped on Fridays, Saturday, or days prior to a
federal holiday.

The GADCC will monitor the Shipping/Receiving Form in the Central
Database and notify PICs when a shipment is in violation of FIND
approved procedures (See Section 5). The Study Coordinator and the
Principal Investigator of the site in question will receive an e-mail
from the GADCC citing the following:

1.  Shipment Date

2.  Federal Express Tracking Number

3.  Barcode ID

4.  Sample Violation or Shipment Violation

5.  Immediate Corrective Action to be Taken

These violations will be recorded in the monthly reports presented to
all members of the Steering Committee, who will also be informed if
corrective action is not taken.

### Replicate (phantom) Samples

An extra set of quality control replicates of whole blood, serum,
plasma, and urine from one study participant will be sent out. Replicate
samples from approximately 10% of the entire FIND study will be selected
for quality control. NCI will generate a list of PIDs that have
specimens in the archive and the amount of each specimen. The GADCC will
eliminate PIDs that do not have enough specimens in the archive to
participate. The remaining specimens will be used to randomly select a
5% sampling for shipment to the Central Phenotyping Laboratory. The NCI
laboratory will use the barcode that contains the PID number and the
tube number as a quality control ID. The NCI will pull the specimens
from their archive, create a shipping form using the quality control ID
as described above, and send the samples to the Central Phenotyping
Laboratory for processing. The Central Phenotyping Laboratory will treat
each sample they receive independent of any previous samples. A new
accession number will be assigned and the usual battery of tests
performed for that specimen. Results of the tests will be reported to
the PICs weekly. The GADCC will receive an electronic report on a
monthly basis from the Central Phenotyping Laboratory. For data analysis
by GADCC, results on each laboratory measurement are matched to the
appropriate participant results using the PID from the barcode ID. GADCC
will generate quality control reports quarterly for review by the
Steering Commitee. The Central Phenotyping Laboratory will be sent the
comparison results on a quarterly basis. These samples will be sent on a
regular basis during the entire study duration. The same procedure will
be utilized for the University of Minnesota Laboratory which performs
additional urine assays for the FIND.

## Sample Receipt at the Central Processing Laboratory (NCI)

### Specimen Identification

Upon arrival at NCI, each specimen is verified by NCI technician via the
GADCC database. If a discrepancy occurs, the NCI technician calls that
specific PIC to resolve the problem.

Procedures for creating and identifying a record for each specimen upon
arrival follows current NCI protocol. This record includes a local
specimen identification number, as well as the FIND ID number. The local
specimen ID number is the linking variable used to update the record for
each specimen (either by direct data transfer from the analytic
instrument or by entry of results from worksheets) after the specimen is
analyzed. It is therefore crucial that care be taken when labeling
specimens with local ID's that the local ID recorded for each specimen
on the data base matches that by which the specimen is actually
identified as it is processed in the lab. Laboratory supervisors
periodically review how this process is carried out and instruct
laboratory technicians in techniques to use to avoid ID errors.

### Maintenance Procedures for Laboratory Equipment

A regular schedule is set up for routine maintenance procedures, with
logbooks kept on their performance. The laboratory supervisor review
these logs on a regular basis to verify that proper maintenance
procedures are being carried out according to the schedule set and that
any special maintenance procedures need are carried out.

## Central Phenotype Laboratory Quality Control (MedStar)

### Specimen Identification

In FIND, blood samples are collected by each PIC and processed by NCI
for shipment to the Central Phenotype Laboratory (MedStar). Procedures
for creating and identifying a record for each specimen upon arrival
follows current MedStar protocol. This record includes a local specimen
identification number, as well as the FIND ID number. The local specimen
ID number is the linking variable used to update the record for each
specimen (either by direct data transfer from the analytic instrument or
by entry of results from worksheets) after the specimen is analyzed. It
is therefore crucial that care be taken when labeling specimens with
local ID's that the local ID recorded for each specimen on the data base
matches that by which the specimen is actually identified as it is
processed in the lab. Laboratory supervisors periodically review how
this process is carried out and instruct laboratory technicians in
techniques to use to avoid ID errors.

MedStar routinely archives all samples at -70^0^C for 30 days after the
initial assays are performed. The archived samples are used to repeat
assays when necessary. GADCC performs bi-weekly evaluation of laboratory
data submitted from MedStar outlying values and systematic patterns. If
outlaying values or systematic patterns are detected, GADCC generates a
list of samples to be repeated and contacts MedStar for repeated assays
to be performed.

## Material Archiving

It is important in handling the FIND frozen blood samples (in the case
of bulk shipments from the PICs) to avoid any unnecessary exposure to
room temperature. Clear procedures for unpacking specimens upon arrival
is set out at NCI to minimize such exposure. NCI archiving has
provisions for (1) prompt detection of power failure or of failure of
freezer to maintain the proper temperature, including both local alarms
and alarm signals to a central security office that will notify
appropriate laboratory personnel if a problem develops after hours; (2)
backup power supplies in the event of power failure; (3) plans for the
use of dry ice to maintain the sample temperature until any problems
with the freezer can be repaired.

## Long Term Consistency of Methods

The laboratory protocols fully describe the measurement methods and
procedures to be used in the FIND. To maintain the long-term
comparability of measurement results throughout the FIND, the same
measurement methods will be maintained throughout the study. If
measurement methods are to be changed, comparison studies must
demonstrate to the satisfaction of the FIND Steering Committee that the
method to be used has been shown to give results fully comparable to the
method used initially. It is important to demonstrate that comparable
results can successfully be achieved in the implementation of the method
in the FIND laboratory, rather than to rely merely on descriptions of
comparisons in the literature, as there are often significant
differences in the results achieved by the same analytic method at
different laboratories. Use of a back-up method in the event of the
temporary failure of the usual FIND method to achieve in-control results
should only be done with the permission of the FIND Steering Committee
after the back-up has already been implemented in the laboratory and
shown to maintain comparable results. In both cases, the experiments to
demonstrate method comparability will be designed jointly by the
laboratory in question and the GADCC.

**Molecular Quality Control**

## Objectives and Goals of the Molecular Quality Control Subcommittee

The goals of the Molecular Quality Control and Assurance Subcommittee
are:

> 1\. To develop protocols and standard operating procedures for
> collection of molecular genetic data from samples ascertained in the
> FIND study with attention to quality control and assurance.
>
> 2\. To plan a genome scan for FIND data with selection of appropriate
> controls and replicate samples.
>
> 3\. To evaluate the molecular results from the genome scan and the
> MALD studies.
>
> 4\. To identify chromosomal regions for follow-up and confirmation of
> linkage results and candidate genes, as per the recommendation of the
> data analysis committee.

5\. To plan positional cloning projects for those regions where initial
and confirmatory evidence for linkage has been obtained

To meet the goals of the molecular quality control and assurance
committee a series of standard operating procedures have been developed.
These are listed below.

## To develop protocols and standard operating procedures for collection of molecular genetic data from samples ascertained in the FIND study with attention to quality control and assurance. 

[]{.mark}

### DNA extraction from lymphoblastoid cell lines

As described in section 8, cell pellets from lymphoblastoid cell lines
will be received at the GADCC laboratory from NCI on a monthly basis,
after the first set of samples have been transformed into cell lines at
the NCI. The receipt of the samples will be scanned into the FIND
database using the FIND barcodes. DNA will be extracted from cell
pellets as per protocol on page 65. Stock DNA will initially be diluted
to 1 microgram/microliter and vials labeled with barcode labels that are
resistant to freezing, moisture and solvent removal. All purified DNA
samples will be diluted to a 200 nanogram/microliter concentration in 10
mM Tris, 5 mM EDTA buffer and stored frozen at --80°C in cryoboxes that
have been barcoded.

### DNA extraction from whole blood or buffy coats

Transformed lymphoblastoid cell lines may be unavailable for DNA
extraction on some patients, although, PICs will be informed of lack of
success in cell line transformation and will be asked to re-draw crucial
samples, if feasible. NCI will inform the GADCC via the reporting fields
in the FIND database that the samples failed to transform, or that
yellow top tubes were unavailable for cell line transformation. In these
cases the archived buffy coat or whole blood sample will be used for DNA
extraction. DNA extraction will be performed using the PureGene Kit
\[Gentra Systems\]. Stock solutions will be diluted to 1
microgram/microliter and vials labeled with barcode labels that are
resistant to freezing, moisture and solvent removal. All purified DNA
samples will be diluted to a 200 nanogram/microliter concentration in 10
mM Tris, 5 mM EDTA buffer and stored frozen at --80°C.

### Assignment of DNA sample to 96-well plates for genotyping

The assignment of a particular DNA sample (10ng/ul) to a particular
address on a specific 96-well Biostar vial will be dependent on whether
the individual participated the MALD study or the individual
participated in the Family Study. Although, samples will generally be
assigned to a plate as collected, the actual assignment will depend on
whether a family qualified for the study and other available family
members. The assignment of a sample will be determined by the FIND
database. For the Family Study, family structures will be retained
inasmuch as allowed by concurrent data collection of family members.
Each DNA plate will also contain the appropriate number of replicate
(N=4) and control CEPH samples (N=4). Using all available samples
replicates will be randomly sampled for each plate while ensuring that
all 5 ethnicites (Caucasian, African American, Mexican American, Pima
Indian and Zuni Indian) are adequately represented. Once a sample is
selected as a replicate it will be removed from further consideration.

### Quality control for DNA concentration

The purity and concentration of the DNA samples will be ascertained
using the spectrophotometer \[Spectromax Plus, Molecular Devices\].
After samples have been diluted to a concentration of 10 ng/ul, a test
PCR reaction will be used to determine if the samples can be amplified
using standard primers. If a particular sample has a DNA concentration
of less than 200 micrograms, the GADCC will ask for additional blood to
be drawn from the individual for genotyping. Samples with DNA
concentrations less than 200 micrograms will not be considered to be
recruited, since a sufficient quantity of DNA is unavailable for
genotyping.

### Shipping of DNA aliquots to the PICs

Each PIC may request that ½ of the archival sample, viable cell-lines or
extracted DNA be returned to them for candidate gene studies. DNA
samples will be shipped to PICs (See Appendix I for addresses) on a
monthly basis. Shipment of samples will be pre-arranged between the
laboratory personnel at the GADCC and the PIC. Samples will be shipped
frozen from Monday through Wednesday and the appropriate personnel
alerted to the arrival of the samples at the PICs. In the event that a
large quantity of DNA is unavailable for a particular sample, a
sufficient amount of DNA from each sample will be retained at the GADCC
for the planned genome scan.

## To plan a genome scan for FIND data with selection of appropriate controls and replicate samples.

### Plan I for the genome scan

1.  In years 2-3.5, an application will be submitted to the Center for
    Inherited Disease Research (CIDR) for a 10cM genome scan for the
    entire FIND study. This application will utilize a two-stage
    approach for the entire sample. First, the DNA sample collected in
    year 2-3.5 (1/4 of the whole sample) will be sent to CIDR for an
    initial genome scan. Next, the sample collected in years 3.5-5 (3/4
    of the whole sample) will be used to replicate the results of the
    initial scan. Thus, samples collected in years 3.5-5 will be shipped
    on an annual basis to CIDR for genome scanning.

2.  CIDR will provide both raw and analyzed data for the study. CIDR
    currently employs 4 CEPH individuals, 4 blind replicates and a
    moving blank on their gels for quality control but is amenable to
    modifying plans to suit the consortiums' needs.

3.  The rationale for the performing an initial genome scan on ¼ of the
    sample is that fine mapping for positive results can be pursued very
    quickly. Four to eight additional markers will be typed in the whole
    sample in regions where significant lod scores are obtained from the
    initial scan (e.g. p-value \<0.05 was observed). The size of the
    interval will determine how to proceed with fine mapping. For an
    interval larger than 3 cMs, additional markers will be typed to
    narrow the interval. For a small interval, candidate genes may be
    tested for mutations and polymorphisms.

4.  The results of the genome scan will be utilized in relationship
    testing and reassigning relationships after assessment of genotyping
    error (see data analysis section).

### Plan II for the genome scan

1.  If the genome scan application is rejected by the CIDR access
    committee because of power considerations or inadequate recruitment,
    a re-submission will be planned within 6 months of the initial
    rejection. Currently, the rate of genotyping error at CIDR is less
    than 1% at CIDR, with a 5% missing data. As a back-up, the molecular
    laboratory at the GADCC will perform the genome scan. In this case,
    only ¼ of the sample will undergo the full genome scan. The whole
    sample will be typed for additional markers where positive lod
    scores are observed. For the first stage scan, 405 equidistant
    markers spanning all human autosomes and the pseudoautosomal region
    of the X chromosome will be used to type each sample. Markers spaced
    at an average distance of 10 cM designed from Co-operative Human
    Linkage Center panels 10, which are composed almost entirely of tri-
    and tetranucleotide repeats with an average heterozygosity of 0.8,
    will be purchased from Research Genetics (Huntsville, AL). The
    current rate of genotyping error at the GADCC laboratory is 5%, with
    10% missing data using the ABI 3700.

2.  Controls and replicates selected for quality control will remain
    identical for both plan I and II.

3.  Markers selected for fine mapping will depend on results obtained
    from the first stage. We anticipate that five to six marker regions
    will "suggest linkage" by chance alone and warrant further
    investigation in order to maintain our desired level of statistical
    power. For each positive region, six to eight markers spaced two cM
    apart on either side of the positive marker will be typed. Further,
    if putative candidate genes lie within 20 cM of the region with
    suggestive linkage, then markers close to or within the gene or the
    candidate gene itself will be typed.

4.  Genotype data will be obtained on an ABI 3700 Sequencer. Allele
    sizes will be assigned using the Genotyper version 2.1 software.
    Data from the Genotyper program will be dispatched to the GADCC
    statistical core for entry into the FIND database and for analysis
    of Mendelian segregation. Data will be analyzed within one week of
    being run. The average size of raw and processed data files obtained
    from this program is about 25-30 megabytes. The data will be
    temporarily stored on the MetroHealth Medical Center server until
    all genotypic information is extracted. The data from each run will
    be compressed and moved for remote storage to a zip disk or CD. A
    version of the raw data files stripped of the large image file and
    compressed to less than half the size will also be stored on the
    Unix Servers at the GADCC. The data in these files will be burned
    onto CDs, media suitable for long-term storage of large amounts of
    data. Each CD will be carefully labeled with dates of the runs,
    markers, and study name, and indexed in the FIND database.

## To evaluate the molecular results from the genome scan and the MALD studies. 

1.  Because data from the genome scan will be accumulated over several
    years, the Molecular Quality control committee along with the Data
    Analysis Committee will analyze the data for measurement drift using
    replicates of samples genotyped in previous years.
    Population-specific allele frequencies will be estimated from the
    data and compared using extant data from public databases such as
    GDB ([www.gdb.org](http://www.gdb.org/)). Raw data and reports for
    each PIC will be made available via web access to the FIND database.
    The progress of the genome scan and the positive evidence for
    linkage will be evaluated in terms of potential candidate genes.

2.  Raw data and results from the MALD studies will also be deposited in
    the FIND database. Similar to methods utilized in the Family Based
    Study, the Molecular Quality control committee along with the data
    analysis committee will record and analyze the MALD genotyping data
    for measurement drift using the quality control plan suggested by
    the MALD centers. The progress of the MALD genotyping and analysis
    will be periodically evaluated in terms of potential candidate
    genes.

## To identify chromosomal regions for follow-up and confirmation of linkage results and candidate genes, as per the recommendation of the data analysis committee.

1.  After the initial genome scan in ¼ of the samples is completed
    (Plan I) and regions that demonstrate positive evidence for linkage
    identified, additional markers in these select regions will be
    identified. These markers will be genotyped in the original sample
    that demonstrated evidence for linkage and in a confirmatory sample,
    if available. Priority of regions for genotyping will be assigned
    based on significance of evidence for linkage. Markers at candidate
    genes selected by the Molecular Access committee and the approved by
    the Steering Committee will also be genotyped in selected samples.

## To plan positional cloning projects for those regions where initial and confirmatory evidence for linkage has been obtained.

1.  If second stage markers confirm evidence for linkage in a particular
    chromosomal region, the evidence in this region will be evaluated
    for an eventual positional cloning project. Additional studies that
    involve reducing the region to a few centimorgans (\<2cMs) will
    first be undertaken. Evaluation of mutations or single nucleotide
    polymorphisms in specific candidate genes are also under the purview
    of this aim.

## Genotyping Protocol for MALD analysis of MA DN Probands and Controls.

DNA samples will be aliquoted in a 96 well format. Working stock 96 well
template containing 1.0 ng DNA/l TE (10 mM Tris/ 1mM EDTA) volume will
be generated using a BIOMEK 2000 robot (Beckman). The working template
trays will be used for robotic aliquoting of 1L into two quadrants of a
384 well microtiter trays that will be used for PCR reactions. A
Multimek (Sagean/Beckman) robotic unit is used for this procedure and
typically at least 4 trays are prepared at the same time at this
station. This procedure is repeated with a second 96 well "working
stock" template so that each quadrant of the 384 well plate contains 96
wells corresponding to one or the other working stock tray. To minimize
any possible run-to-run bias each 384 well plate will contain two
quadrants from a proband 96 well working stock and two quadrants from a
control 96 well working stock.

All PCR reactions will be performed in 384 well PCR trays. Typically 3
markers will be multiplexed for each PCR reaction and PCR reaction mixes
are robotically dispensed into two quadrants of the 384 well PCR tray in
which template DNA has been predispensed. This typically repeated so
that a total of 8 different PCR reaction mixes that are dispensed at the
same time into 4 different 384 well trays. Each of the primer sets are
designed to have compatible PCR products (by size or different flours).
When additional multiplexing of compatible products is possible a total
of upto 12 different PCR reaction mixes can be performed at the same
time without exceeded the capacity of the robotic Biomek robotic station
or thermocyclers. The final 5 μl PCR contains 2.65 μL H2O, 0.5 μl PCR
buffer, 0.7 μl of 2.5 mM dNTPs, 0.1 μL of 10 μM mix of forward and
reverse primers and 0.05μl Taq polymerase.

The primers pairs amplify genomic DNA segments of \~100 to 400 bps that
contain specific MALD microsatellite or insertion/deletion (I/D)
polymorphisms. The forward primer is conjugated with one of three ABI
3700 sequencer compatible fluorescent dyes (FAM, HEX or NED). These are
stored in light protected boxes at --20^0^C prior to PCR setup. For
microsatellite polymorphisms most reverse primers will be synthesized to
contain tails to minimize the plus A problem that is especially an issue
for dinucleotide repeat markers.

To accomplish the PCR setup, a master-mix sufficient for 384 PCRs
containing the entire reaction mix except for the template is aliquoted
and placed in a Biomek test-tube station. Using one of our standard
programs the Biomek 2000 accurately dispenses the required 4 μL volume
into the specified two 96 well quadrants of the appropriate 384 well PCR
tray. The PCR tray is then sealed (Micro Amp Clear Adhesive Film),
vortexed briefly and then centrifuged.

The completed and mixed 384 well PCR trays are then placed in the
thermocyclers. For the typical protocol (as described above), 4 tray
PCRs are performed using two of our three dual 384 well thermocyclers
(Gene Amp PCR System 9700, PE Biosystems). For most reactions a standard
PCR program similar to the Marshfield protocol is used for both
microsatellite and I/D polymorphisms (95^0^C X 3 min, 95^0^C X 45 sec,
58^0^C X 1.5 min, 72^0^C X 45 sec for 32 cycles). Products are either
stored at --20^0^C (for upto 2 weeks) at this stage or are prepared for
3700 loading.

After the PCR reaction has been completed and within 12 hours of
initiating an ABI 3700 sequencer run, a Multimek robot is used to mix
the compatible PCR products and set up loading trays. As indicated above
the reaction templates are set up so that a given quadrant is typically
matched in four trays by identical templates and PCR products that are
size/fluor compatible for multiplexing. The four trays can therefore be
combined by simply retaining the same quadrant position in a 384 loading
tray. For this tray, 0.5 μl of each PCR product from each of the four
PCR trays is mixed using one of our laboratory's standard Multimek
programs. The loading tray is then prepared by adding 4 μl of loading
mixture. The loading mixture contains 90 ml of 3700 BAL ROX standard
(Bioventures)/4000 μL of low conductivity formamide (Amersham). This
size standard contains fragments in 20 bp intervals throughout the size
range 80 to 400 bp plus 70, 90 and 190 bp fragments.

Two 384 well completed loading trays are then placed in the ABI 3700 (PE
Biosystems) for gel capillary size separation. This automated sequencer
is run using the standard ABI operating protocol with the POP6 run
polymer. After the sequencer run has been completed the run files are
transferred from the 3700 dedicated computer to another PC (Dell,
Pentium III, 750 mhz) . The Genescan Analysis software is used to size
the DNA fragments based on the sizes of the size standard peaks. This
"raw data" consisting of DNA fragment sizes for each typed individual is
then imported into the Genotyper software program (PE Biosystems) in
which templates have been created for each marker, specifying marker
name, range of allele sizes and fluorescent dye label and peak height
thresholds. This information is used by the software to generate
genotypes (to 2 decimal places) for each individual typed and this is
displayed in tabular and graphical forms. Each genotype is then manually
reviewed to minimize errors in the labeling of alleles by the software.
Visual confirmation of each individual peak height is performed by
careful review of the electrophoretic profiles. A final table of
genotype results is then saved as an Excel file.

Allele binning will then be performed using the analysis tool package in
EXCEL (97 or later versions) prior to exporting data for specific
statistical analyses.

### Quality Control:

To enable checking the quality and reproducibility of the genotyping
each of the 96 well templates will contain 4 replicate samples and 4
CEPH controls (as provided by the FIND GADCC). The known positions of
the CEPH controls will be used to establish consistency in allele
assignments between sets run and genotyped on different days. The
replicate samples (with sample positions blinded to technical staff)
will be used to determine the concordance rate for individual marker
typing. If the marker concordance is less than 95% the marker will be
re-genotyped carefully examining the electophoretograms for artifactual
peaks and reviewing the binning criteria. If the marker concordance is
still less than 95% the marker will either be discarded or will be
retyped starting from the initial PCR step.

## Genotyping Protocol for MALD analysis of African Americans.

DNA samples will be aliquoted into 4.5 ml cryogen type tubes. Working
stock templates containing 1.0 ng DNA/ul will be generated using a
Packard Multiprobe II robot. The 300 ul of the working template stocks
will then be robotically aliquoted into deep-dish 384-well plates. Those
daughter plates will be transferred into 384-well PCR granddaughter
plates hydra containing 5ng/well with a Robbins 384 well hydra. Plates
are then dried overnight in a laminar flow hood, sealed with tape and
stored for up to six months at --20 C for subsequent PCR analysis.

The NCI will perform these analyses using state of the art technologies
that are constantly being evaluated and implemented to improve the
genotyping process. If 1532-well PCR systems are commercially available
and reliable at the time the MALD analysis begins those will be used in
the PCR analysis using a Robins 384 Tango hydra system for aliquoting
and pooling. Subsequent discussions of these plans will assume 384-well
density even though the higher 1532-well density will hopefully be
available in the next one to two years.

All PCR reactions will be performed in 384 well PCR trays. PCR reaction
mixes are robotically dispensed into the 384 well PCR trays in which
template DNA has been pre-dispensed and rehydrated. This is typically
repeated so that many different PCR reaction mixes that are dispensed at
the same time into different 384 well trays. Using the newly released
five-dye system from Applied Biosystems, each of the primer sets will be
designed to have compatible PCR products (by sizes up to 500 bp and four
different flours). Up to twenty loci can be independently amplified,
pooled and then analyzed.

Fragments will be size separated on Applied Biosystems 3100 sequencers,
or their successors depending on availability. Electropherograms that
are produced by the sequencers will be analyzed in Gene Mapper (Applied
Biosystems) to bin alleles for subsequent analysis. These data will be
deposited into a database at the NCI and analyzed for association
between the FIND phenotypes and MALD genome scan markers.

### Quality Control:

To enable checking the quality and reproducibility of the genotyping
each of the 384 well templates will contain 8 replicate samples used in
Dr. Michael Smith's laboratory in all 384 well plate genotyping
applications. The internal duplicates will be used to establish the
consistency of genotyping in the laboratory. Internal blinded replicate
samples will be used to determine error rates and determine which
markers must be retyped or redesigned.

**Quality Control Procedure**

> A random set of 400 individuals from the family study and 400
> individuals from the MALD study will be selected and sent to an
> external laboratory for DNA profiling. DNA for each participant
> selected will be extracted from a buffy coat and from the b-cell
> pellet. The DNA will be quantified and set up in a 96 well plate. All
> samples will have a unique barcode ID and are anonymized to the
> outside laboratory. The DNA will then be DNA diagnostics for
> profiling. The results will be reported on the GADCC every two weeks
> until all samples have been profiled. The GADCC will review the
> profiling results and report their findings to the Steering Committee
> and NIH.