HLA-DRB BigDye™ Terminator Sequencing

HLA-DRB BigDye™ Terminator Sequencing
HLA-DRB
BigDye™ Terminator Sequencing-Based Typing Kit
Protocol
For Research Use Only. Not for use in diagnostic procedures.
© Copyright 2001, Applied Biosystems
For Research Use Only. Not for use in diagnostic procedures.
Printed in the U.S.A.
Notice to Purchaser: Limited License
The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences for the HLA
DRB gene, except as stated herein. For research and development, a license under U.S. Patents 4,683,202, 4,683,195, 4,965,188 and
5,075,216 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche Ltd. (“Roche”), has an upfront fee component and a running-royalty component. The purchase price of the HLA DRB BigDye™ Terminator SequencingBased Typing Kit (P/N 4305213, 4305026 or 4305027) includes limited, nontransferable rights under the running-royalty
component to use only this amount of the product to practice the Polymerase Chain Reaction (“PCR”) and related processes
described in said patents for the research and development activities only of the purchaser and only when this product is used in
conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must
be obtained by the end user in order to have a complete license to use this product in the PCR process for research and development
activities. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an
Authorized Thermal Cycler. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting
the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular
Systems, Inc., 1145 Atlantic Avenue, Alameda, California, 94501. No general patent or other license of any kind other than this
specific right of use from purchase is granted hereby.
Notice to Purchaser About Limited License
This kit (reagent) is sold pursuant to a limited sublicense from Amersham International plc under one or more U.S. Patent Nos.
5,498,523, 4,994,372, U.S. Patent Application Serial Nos. 08/324437, 08/337615, and corresponding foreign patents and patent
applications. The purchase of this kit (reagent) includes a limited non-exclusive sublicense (without the right to resell, repackage,
or further sublicense) under such patent rights to use this reagent for DNA sequencing or fragment length analysis solely with an
Applied Biosystems commercial automated sequencing machine or other authorized DNA sequencing machines that have been
authorized for such use by Applied Biosystems Division of the PE Corporation, or for manual DNA sequencing. No license is hereby
granted for the use of this kit, or the reagents therein, in any other automated sequencing machine. Such sublicense is granted solely
for research and other uses that are not unlawful. No other license is granted expressly, impliedly, or by estoppel. For information
concerning the availability of additional license to practice the patented methodologies, contact: Amersham Life Science, Inc., Vice
President, Regulatory Affairs, P.O. Box 22400, Cleveland, Ohio 44122. Patents are pending in countries outside the United States.
ABI, Applied Biosystems, BigDye, MatchMaker, MatchTools, MTNavigator, and POP-6 are trademarks and ABI PRISM and its
design, MicroAmp, and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and
certain other countries.
AmpliTaq, AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems, Inc.
All other trademarks are the sole property of their respective owners.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
SSP-PCR Amplifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Sequencing SSP-PCR Fragments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Allele Assignment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Instrument Platforms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Materials and Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Reagents Included. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Reagents and Equipment Not Included. . . . . . . . . . . . . . . . . . . . . . . . . .3
Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
To Reach Us On the World Wide Web . . . . . . . . . . . . . . . . . . . . . . . . . .7
To Reach Us by Telephone or Fax . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . .7
Documents-on-Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
To Reach Us by E-Mail. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Regional Offices, Sales and Service . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Blood Collection and DNA Sample Preparation . . . . . . . . . . . . . . . . . . . . . . .14
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Low-resolution SSP-PCR DRB Typing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Low-resolution SSP-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Thermal Cycling Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Analysis of Low-resolution PCR Amplifications . . . . . . . . . . . . . . . . . . . . . .20
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Low-resolution Results as Control Reactions. . . . . . . . . . . . . . . . . . . .22
i
Forward and Reverse Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Performing Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Purifying Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Precipitating Sequencing Reactions in a MicroAmp 9600 Tray . . . . . 25
Precipitating Sequencing Reactions in Single Tubes . . . . . . . . . . . . . . 26
Spin Column Purification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Rehydrating the Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Using the Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Performing Codon 86 Group-Specific Sequencing. . . . . . . . . . . . . . . . . . . . . 29
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Performing Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Recommendations for ABI PRISM 310 Genetic Analyzer Users . . . . . . . . . . 31
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Capillary Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Preparing and Loading Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Recommendations for Sample Naming . . . . . . . . . . . . . . . . . . . . . . . . 33
Setting up Default Sample Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Sample Sheet Menu choices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Filling in the Injection List. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Signal Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Performing Fluorescent Signal Analysis . . . . . . . . . . . . . . . . . . . . . . . 35
Recommendations for ABI PRISM 377 DNA Sequencer Users . . . . . . . . . . . 36
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Gel Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Preparing and Loading Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Recommendations for Sample Naming . . . . . . . . . . . . . . . . . . . . . . . . 37
Entering default file names for the Sample File. . . . . . . . . . . . . . . . . . 37
Sample Sheet Menu choices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Loading Sample on the ABI PRISM 377. . . . . . . . . . . . . . . . . . . . . . . . 38
ABI PRISM 377 Run Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Performing Fluorescent Signal Analysis . . . . . . . . . . . . . . . . . . . . . . . 39
ii
Assignment of HLA-DRB Allele Types to Sequence Files . . . . . . . . . . . . . . .41
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Using MatchTools Software for Preliminary Analysis . . . . . . . . . . . . .41
HLA-DRB Allele Libraries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Using MTNavigator Software for Editing . . . . . . . . . . . . . . . . . . . . . .42
Using MatchTools Software for Final Analysis . . . . . . . . . . . . . . . . . .43
Appendix A. HLA-DRB Typing Results Template . . . . . . . . . . . . . . . . . . .44
Sample Form. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
Appendix B. HLA-DRB Comparison Table . . . . . . . . . . . . . . . . . . . . . . . . .45
Low Resolution SSP-PCR Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Appendix C. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
MatchTools Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
Anomalies Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
IUB Codes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Appendix D. Instrument (Matrix) Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60
Multicomponent Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60
Making a Matrix from a Sample File . . . . . . . . . . . . . . . . . . . . . . . . . .61
Run the Sequencing Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
Make Copies of the Standard Run . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
View Matrix Run Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
Generate the Terminator Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
Complete the Other Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Evaluate Matrix Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
iii
Introduction
Overview IMPORTANT
This kit should be used in accordance with guidelines
established by the Committee for Quality Assurance and Standards of the
American Society for Histocompatibility and Immunogenetics.1
The genetic complexity of the DRB region of the human major
histocompatibility complex (MHC) has required development of
molecular typing techniques with increasing levels of resolution.
Methods such as Polymerase Chain Reaction–Sequence-Specific
Oligonucleotide Probe (PCR-SSOP) or Sequence-Specific
Primer–PCR (SSP-PCR) are not always able to discriminate among the
approximately 200 recognized alleles at DRB1.
To address this need, Applied Biosystems has developed a system that
combines a low-resolution SSP-PCR followed by high-resolution allele
typing using automated DNA sequencing.
SSP-PCR The low resolution SSP-PCRs are based on allele group-specific motifs
Amplifications in the first hypervariable region of exon 2. The following allele groups
and genes are amplified with the eleven specific PCR mixes provided:
♦
DR1
♦
DR7
♦
DRB3
♦
DR2
♦
♦
DR3/11/6
♦
DR8/12
♦
DRB4
DR9
♦
DRB5
♦
DR4
♦
DR10
In addition to the group-specific amplification mixes, a control mix is
also provided that amplifies all DRB alleles and genes (DRB*ALL). This
mix can be used as either a positive or negative amplification control.
These PCRs have been optimized for use with AmpliTaq Gold® DNA
Polymerase to ensure specific and efficient reactions.
1.
1996 ASHI Standards for Histocompatibility Testing. American Society for
Histocompatibility and Immunogenetics, 1996 Membership Directory, pp. 17–28.
1
Sequencing The positive amplification reactions from the SSP-PCR are used as
SSP-PCR sequencing templates to generate the high resolution allele typing
Fragments. information. The PCR and sequencing protocols enable direct
sequencing with no PCR purification steps. The design of the
amplification primers allows sequencing of both strands from a single
amplification fragment. The BigDye™ terminator cycle sequencing
chemistry, provided in the Ready Reaction format, has been optimized
for ease of use and high throughput with less technician time.
Allele Assignment The final step in the analysis procedure is to perform the allele
assignment using MatchTools™ Software and MTNavigator™ Software
provided in the MatchMaker™ Allele Identification Software package.
These programs work together to assign alleles and to allow manual
review or editing of the sequencing data.
Instrument The protocols provided in this document were optimized using the
Platforms GeneAmp® PCR System 2400 and GeneAmp® PCR System 9600
Thermal Cyclers. Specific instructions are given for the HLA-DRB kit
reagents to generate data on the ABI PRISM® 310 Genetic Analyzer,
and the ABI PRISM® 377 DNA Sequencer. The protocol identifies certain
steps where the protocol varies between the instruments. For more
detailed instructions on using the instruments, refer to the appropriate
instrument user’s manual.
IMPORTANT
This kit is not designed for use with the ABI™ 373 DNA
Sequencer or the ABI 373 DNA Sequencer with the XL Upgrade.
2
Materials and Equipment
Reagents Included The HLA-DRB BigDye™ Terminator Sequencing-Based Typing Kit
includes the reagents listed below. One kit is sufficient for generating
complete HLA-DRB typings for 48 purified DNA samples.
♦
♦
HLA-DRB Amplification Module (for amplification of 48 purified
DNAs)
–
One tube of AmpliTaq Gold DNA Polymerase (50 µL, 5 U/µL)
–
12 tubes of PCR Mixes, one for each SSP-PCR
HLA-DRB Sequencing Module (sufficient for sequencing
96 templates generated with the HLA-DRB Amplification Module)
–
Two tubes of HLA-DRB Forward Sequencing mix
–
Two tubes of HLA-DRB Reverse Sequencing mix
–
Two tubes of DNA Diluent Buffer
Reagents and In addition to the reagents supplied in this kit, other items are required
Equipment Not depending on which sequencing instrument is used. Refer to the
Included individual instrument manuals for the specific items needed. Many of
the items listed are available from major laboratory suppliers (MLS)
unless otherwise noted. Equivalent sources may be acceptable where
noted.
Control and Loading Buffer Module
The Control and Loading Buffer Module (P/N M0028) is sold separately
and contains the following:
♦
Two tubes of human DNA, male (DRB1*0301/1001, DRB3*0202)
(10 ng/µL, 100 µL each tube)
♦
Five tubes of recrystallized formamide (0.5 mL each)
♦
One tube of blue dextran/EDTA solution (1.0 mL)
Table 1.
Reagents Supplied by the User
Reagent
Source
ABI PRISM dRhodamine Matrix Standards Kit
Applied Biosystems
(P/N 403047)
Agarose gel loading buffer
MLS
3
Table 1.
Reagents Supplied by the User
(continued)
Reagent
Source
Agarose, molecular biology grade
MLS
Boric acid
MLS
Deionized water
MLS
Disodium ethylenediaminetetraacetic
acid dihydrate (Na2EDTA•2H2O)
Sigma (P/N E4884) or
Gibco BRL (P/N SS7SUA)
DNA size ladders, 1 µg/µL,
100-bp or 250-bp
Gibco BRL
(P/N 15628-050, 10596-013)
Ethanol (absolute)
MLS
Ethanol (anhydrous)
Kodak IBI (P/N IB15720)
Ethidium bromide
Sigma (P/N E1510) or MLS
Sodium acetate, 3 M, pH 4.6
Applied Biosystems
(P/N 400320)
Tris base, molecular biology grade
MLS
Tris-HCl, molecular biology grade
MLS
For the ABI PRISM 310 Genetic Analyzer Only
10X Genetic Analyzer Buffer with EDTA
Applied Biosystems
(P/N 402824)
Performance Optimized Polymer 6 (POP-6™)
Applied Biosystems
(P/N 402837)
For the ABI PRISM 377 DNA Sequencer Only:
4
Ammonium persulfate
Kodak IBI (P/N IB70080)
Long Ranger Gel Solution, 50%
FMC (P/N 50611)
Long Ranger Singel pack
Type: 377-36 cm WTR
FMC (P/N 40691)
N,N,N´,N´-Tetramethylethylenediamine
(TEMED)
Kodak IBI (P/N IB70120)
Urea
Kodak IBI (P/N IB72064)
Table 2.
Equipment Supplied by the User
Item
Source
ABI PRISM 377 DNA Sequencer, or
ABI PRISM 310 Genetic Analyzer
Applied Biosystems
ABI PRISM® DNA Sequencing Analysis
Software, v. 2.1.1, v. 2.1.2, or v. 3.0
Applied Biosystems
GeneAmp PCR System 2400 or 9600
Applied Biosystems
Heating block
MLS
MatchMaker™ Allele Identification Software, v.
1.0
(includes MatchMaker™, MatchTools™ and
MTNavigator™ software)
Applied Biosystems
(P/N M0024)
MicroAmp® 2400 or 9600 Base
Applied Biosystems
(P/N N801-0531, N801-5531)
MicroAmp® 2400 or 9600
Tray/Retainer Set
Applied Biosystems
(P/N N801-0530, N801-5530)
MicroAmp® 2400 or 9600 Full Plate Cover
Applied Biosystems
(P/N N801-0550, N801-5550)
MicroAmp® caps, 8 or 12/strip
Applied Biosystems
(P/N N801-0535, N801-0534)
MicroAmp® Optical 96-Well Reaction Plate
Applied Biosystems
(P/N N801-0560)
MicroAmp® Reaction Tubes, 0.2-mL,
or strip tubes
Applied Biosystems
(P/N N801-0533, N801-0580)
Multichannel pipettor
MLS
Pipettes and tips
MLS
Spin Column, Centri-Sep™, 1-mL
Princeton Separations
(P/N CS-901)
Outside North America:
Applied Biosystems
(P/N 401763, P/N 401762)
Tabletop centrifuge,
with 96-well microtiter tray adaptor
MLS
Vortexer
MLS
5
Table 2.
Equipment Supplied by the User
Item
(continued)
Source
For the ABI PRISM® 310 Genetic Analyzer Only
ABI PRISM 310 Genetic Analyzer capillary,
labeled with a green mark (Lt = 47 cm,
Ld = 36 cm, i.d. = 50 µm)
Applied Biosystems
(P/N 402839)
ABI PRISM 310 Genetic Analyzer Sample Tubes, Applied Biosystems
0.5-mL
(P/N 401957)
ABI PRISM 310 Genetic Analyzer septa for
0.5-mL sample tubes
Applied Biosystems
(P/N 401956)
ABI PRISM 310 Genetic Analyzer Buffer Vials,
4.0-mL
Applied Biosystems
(P/N 401955)
Genetic Analyzer Retainer Clips
Applied Biosystems
(P/N 402866)
Genetic Analyzer Septa Strips, 0.2 mL Tube
Applied Biosystems
(P/N 402059)
Glass syringe, 1.0-mL
Applied Biosystems
(P/N 604418)
Safety Information For information on the safe operation of the ABI PRISM 310 Genetic
Analyzer, refer to the ABI PRISM 310 Genetic Analyzer Site Preparation
and Safety Guide.
For information on the safe operation of the ABI PRISM 377 DNA
Sequencer, refer to the ABI PRISM 377 DNA Sequencer Safety
Summary.
6
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9
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c. Call 1-800-487-6809 from a touch-tone phone a
second time.
d. Press 2 to order up to five documents and have
them faxed to you.
by phone from
outside the
United States or
Canada
a. Dial your international access code, then
1-858-712-0317, from a touch-tone phone.
Have your complete fax number and country code
ready (011 precedes the country code).
b. Press 1 to order an index of available documents
and have it faxed to you. Each document in the
index has an ID number. (Use this as your order
number in step “d” below.)
c. Call 1-858-712-0317 from a touch-tone phone a
second time.
d. Press 2 to order up to five documents and have
them faxed to you.
10
To Reach Us by Contact technical support by e-mail for help in the following product
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13
Blood Collection and DNA Sample Preparation
Procedure ! WARNING ! BIOHAZARD. Blood samples have the potential to
transmit infectious diseases. Follow the latest guidelines published by the
Centers for Disease Control (CDC) and National Institutes of Health (NIH)
concerning the principles of risk assessment, biological containment,
and safe laboratory practices for activities involving clinical specimens.
These principles can be found in the U.S. Department of Health and
Human Services (HHS) publication, Biosafety in Microbiological and
Biomedical Laboratories (publication number 93-8395, stock number 017040-523-7). The biosafety Level-2 containment elements are consistent
with the Occupational Safety and Health Administration (OSHA)
requirements contained in the HHS OSHA Bloodborne Pathogen Standard
29 CFR, part 1910.1030.
To collect and prepare blood samples :
Step
Action
1
Collect a sample of venous blood into an ACD-A, EDTA, or
heparin tube.
2
Prepare genomic DNA following appropriate blood handling
procedures.
The following methods have been shown to yield high-quality, highmolecular-weight genomic DNA directly from samples of fresh
blood:
♦
QiaAmp Blood Kit (Qiagen, P/N 29104)
♦
PureGene (Gentra, P/N D5000)
♦
Isoquick Nucleic Acid Extraction Kit (Orca, P/N MXT-020-100)
♦
Manual salting outa,b
These DNA purification kits provide DNA that works well with the
Applied Biosystems HLA typing kits. Other methods may work, but
should be validated before routine use.
14
To collect and prepare blood samples
Step
(continued):
Action
3
Prepare DNA dilution buffer (10 mM of Tris-HCl, 0.1 mM of EDTA,
pH 8.0).
4
Quantitate and resuspend purified DNA in DNA dilution buffer to a
concentration of 10 ng/µL.
5
Store at 2–6 °C.
a. Miller, S. A., Dykes, D. D. and Polesky, H. F., 1988. “A Simple Salting Out Procedure for
Extracting DNA from Human Nucleated Cells.” Nucleic Acids Research 16: 1215.
b. Proceedings of the Eleventh International Histocompatibility Workshop and Conference,
Yokohama, Japan, November 6–13, 1991.
15
Low-resolution SSP-PCR DRB Typing
Overview The low resolution DRB typing is based on the SSP-PCR results
generated with the reagents in the HLA-DRB Amplification Module. For
each DNA sample to be tested, 12 separate PCR amplifiications are
performed.
IMPORTANT
These manipulations must be performed in an environment
free of contamination by human DNA or PCR amplicons, such as a separate
clean room or biological containment hood. Vortex all reagents briefly before
using.
Low-resolution
To perform low-resolution SSP-PCR
SSP-PCR
Step
1
Action
Prepare samples at room temperature as follows:
IF preparing…
THEN…
Allele Group-specific
Reactions
a.
Add 1.0 µL AmpliTaq Gold
DNA Polymerase (stock
solution at 5 U/µL) to 40 µL
genomic template (10 ng/µL).
b.
Add 10 µL of each Allele
Group-specific PCR Mix to
0.2-mL MicroAmp® PCR
tubes.
c.
Add 2.5 µL of genomic
template/AmpliTaq Gold DNA
Polymerase mixture to the
PCR mixes.
(DRB1G1, DRB1G2,
DRB1G3/11/6, DRB1G4,
DRB1G7, DRB1G8/12,
DRB1G9, DRB1G10, DRB3,
DRB4, and DRB5)
Positive Control Reactions
(DRB*ALL)
16
Add 2.5 µL of the genomic
template/AmpliTaq Gold DNA
Polymerase mixture to a 0.2-mL
MicroAmp PCR tube containing
10 µL of the DRB*ALL PCR mix.
To perform low-resolution SSP-PCR
Step
(continued)
Action
Negative Control
Reactions
(DRB*ALL)
Combine 1.0 µL of AmpliTaq Gold
DNA Polymerase with 40 µL DNA
Dilution Buffer.
Add 2.5 µL of DNA Dilution
Buffer/AmpliTaq Gold DNA
Polymerase mixture to a 0.2-mL
MicroAmp PCR tube containing
10 µL of DRB*ALL PCR Mix.
Note
Using the DRB*ALL PCR as a positive or negative control
is up to the individual user. If detecting contamination is critical,
then using the DRB*ALL as a negative control is appropriate. If it is
important to ensure that the DNA sample will in fact work in the
amplification reactions, then it may be more appropriate to use the
DRB*ALL as a positive control.
Note
An indicator dye may be used by adding 1.2 µL of a
25 mM solution of cresol red dye to the genomic template/AmpliTaq
Gold mixture. The genomic template/AmpliTaq mixture has been
added to the PCR reactions if the final solutions have slight red
tints.
2
Place the tubes in a 96-well or 24-well tube tray/retainer depending
on the thermal cycler to be used.
A suggested tube arrangement for four DNA specimens in a
96-well tray is shown below. Each row corresponds to a different
sample. The first eleven columns in each row contain Allele Groupspecific PCR Mix, genomic template, and AmpliTaq Gold DNA
Polymerase. The twelfth column contains DRB*ALL PCR Mix as
either a positive or negative control.
17
To perform low-resolution SSP-PCR
DRB*ALL
DRB5
DRB4
DRB3
DRB1G10
DRB1G9
DRB1G8/12
DRB1G7
DRB1G4
DRB1G3/11/6
DRB1G2
Action
DRB1G1
Step
(continued)
Samples
18
3
Spin tubes briefly in a tabletop centrifuge fitted with a 96-well or
24-well tray support to collect their contents at the bottoms of the
tubes.
4
Seal the tubes with MicroAmp Caps or cover them with a
MicroAmp 9600 or 2400 Full Plate Cover.
5
Place the tray in the thermal cycler (GeneAmp PCR System 9600,
or 2400).
Thermal Cycling For either the GeneAmp PCR System 9600 or 2400, set the reaction
Conditions volume to 12 µL.
Program the thermal cycler to:
Step
1
2
3
Action
Do 1 cycle of the following to activate AmpliTaq Gold:
♦
Rapid thermal ramp to 95 °C
♦
95 °C for 10 min.
Repeat the following for 36 cycles:
♦
96 °C for 20 seconds
♦
Rapid thermal ramp to 65 °C
♦
65 °C for 30 seconds
♦
Rapid thermal ramp to 72 °C
♦
72 °C for 30 seconds
Do 1 cycle of the following:
Rapid thermal ramp to 99 °C
99 °C for 10 min.
4
5
Do 1 cycle of the following:
♦
Rapid thermal ramp to 4 °C
♦
4 °C forever
After amplification, store PCR products at 2–6 °C until needed.
19
Analysis of Low-resolution PCR Amplifications
Protocol
To analyze low-resolution PCR amplifications:
Step
1
Action
Prepare a 2% agarose gel containing 1X TBE buffer and 0.5 µg/mL
ethidium bromide.
! WARNING ! CHEMICAL HAZARD. Ethidium bromide is a
known mutagen. It can change genetic material in a living cell.
Before using ethidium bromide as a stain, read the
manufacturer’s MSDS, which gives information on physical
characteristics, hazards, precautions, first-aid, spill clean-up,
and disposal procedures. Wear appropriate protective
eyewear, clothing, and gloves.
20
2
Combine 7.5 µL of each PCR amplification reaction with 3 µL of
agarose gel loading buffer.
3
Load 10 µL on the agarose gel.
4
Load in one lane 0.5 µg of BRL 100-bp or 250-bp size ladder in
loading buffer.
5
Run the gel in 1X TBE at 10 V/cm until the fragments in the size
ladder resolve. For example, a 100 mL, 10 ¥ 5 cm submarine gel
run at 150 V for 30 minutes is sufficient.
To analyze low-resolution PCR amplifications:
Step
(continued)
Action
DRB*ALL
DRB5
DRB4
DRB3
DRB1G10
DRB1G9
DRB1G8/12
DRB1G7
Compare the results with the table in Appendix B on page 45.
DRB1G4
8
DRB1G3/11/6
Record the electrophoresis results on a form like the one in
Appendix A on page 44.
DRB1G2
7
DRB1G1
Photograph the resulting electrophoresis pattern. The photograph
below shows that for this sample, the DRB1G4, DRB1G7, and
DRB4 alleles are present.
MW
6
Note
An amplification reaction is considered positive if an SSPPCR fragment has an intensity comparable to either the 600-bp
fragment of the 100-bp ladder, or the 1000-bp fragment of the
250-bp ladder.
21
Low-resolution Considerations to keep in mind while reviewing the low resolution
Results as Control results include the following:
Reactions ♦ Only one or two of the DRB1 PCRs should be positive. If three or
more DRB1 amplifications are positive, then contamination or a
PCR problem may exist. If none is positive, then there may be a
problem with the sample, the reagents, and/or the thermal cycler.
♦
The positive DRB1 PCRs should be consistent with the DRB3,
DRB4, and DRB5 results based on known linkage patterns. These
patterns are detailed in the table in Appendix B on page 45. For
example, refer to the figure in step 6 on page 21. Note that
DRB1G4 and DRB1G7 are positive and that DRB4 is also positive.
♦
Known exceptions to these rules include the few alleles that do not
amplify in the expected allele groups as listed below.
DRB1G8/12
DRB1G4
1105
1122
1317
1410
1404
1415
1428
1431
♦
Other DRB haplotypes may have an unusual amplification pattern.
If a combination is observed that is not consistent with the table on
page 45, then:
–
Record your observations.
–
Check your results carefully after sequencing and allele
assignment.
–
Consult the latest allele sequence layouts and scientific
literature.
For example, some DR2 alleles are on haplotypes that lack the
DRB5 gene.
22
Forward and Reverse Sequencing
Overview High resolution allele typing is based on sequencing the positive SSPPCR reactions. This provides sequence for exon 2 in both the forward
and reverse orientations.
The following section describes the steps involved in preparing forward
and reverse sequencing reactions from a single SSP-PCR amplicon.
There is no requirement for PCR purification in this procedure; the
amplicon is simply diluted 1:3 in DNA Diluent buffer or distilled water.
Depending on the number of positive reactions in the low resolution
step, the total number of Forward and Reverse sequencing reactions
will be between two and eight. The sequencing is performed using
Ready Reaction mixes that include the sequencing primer and
BigDye™ Terminator Mix.
Performing Note Use MicroAmp Reaction Tubes or a MicroAmp Optical 96-Well
Sequencing Reaction Plate.
Reactions Note Perform thermal cycling using the correct method. Set the sample
volume to 10 µL.
To perform the sequencing reactions:
Step
1
Action
Dilute PCR product 1:3 in DNA Diluent.
For sufficient sample to sequence in both directions, add 2 µL of
PCR product to 6 µL of DNA Dilution Buffer.
2
To separate tubes/plate wells, add 8 µL of each of the Ready
Reaction Mixes.
3
To each of the Ready Reaction Mixes in the tubes/plate wells, add
2 µL of diluted PCR product.
4
Seal the tubes with MicroAmp caps or cover the plate with a
MicroAmp 2400 or 9600 Full Plate Cover.
5
Centrifuge briefly in a benchtop centrifuge to combine the PCR
product and Ready Reaction Mix at the bottom of the tubes/plate
wells.
6
Place the tubes/plate in a thermal cycler.
23
To perform the sequencing reactions:
Step
7
8
9
(continued)
Action
Do 1 cycle of the following:
♦
Rapid thermal ramp to 96 °C
♦
96 °C for 10 seconds
Repeat the following for 20 cycles:
♦
96 °C for 10 seconds
♦
Rapid thermal ramp to 50 °C
♦
50 °C for 10 seconds
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 2 minutes
Rapid thermal ramp to 4 °C and hold until ready to purify the
sequencing reactions.
Purifying Following cycle sequencing, the reactions are purified and concentrated
Sequencing to remove unincorporated dye terminators that can appear as artifacts
Reactions in the sequencing data.
Three methods have been validated for recovering the sequencing
ladders following the cycle sequencing reactions. For ease of use as
well as increased throughput, we recommend the ethanol precipitation
procedure using the 96-well format. For those laboratories that do not
have access to a bench-top centrifuge fitted with a 96-well tray adaptor,
two alternative methods can be used: a single tube ethanol precipitation
method or a Centri-Sep™ spin column procedure.
24
Precipitating
Sequencing
Reactions in a
MicroAmp 9600
Tray
Perform this step in the same tubes/plate wells as the cycle sequencing
reaction. This helps minimize the number of sample transfers and
potential sample mix-ups.
Note
Use the MicroAmp 9600 Base and Tray Retainer to hold the tubes.
Note
If multiple samples are processed in a 96-well format, using a
multichannel pipettor can simplify precipitation.
IMPORTANT
Perform the steps below without interruption and in the exact
order they are given. If the precipitated reactions sit between steps, the signal
strength will be decreased.
IMPORTANT
Remove the ethanol completely from the precipitated
reactions as it may contain unincorporated terminators and dye degradation
products.
To perform ethanol precipitation:
Step
1
Action
Prepare a fresh mix:
1.0 mL anhydrous ethanol (EtOH)
40 µL 3M sodium acetate (NaOAc) pH 4.6
2
To each sequencing reaction add 25 µL of the EtOH/NaOAc mix.
3
Replace the strip caps or full plate cover and mix thoroughly by
vortexing or inversion.
4
In a centrifuge fitted with a 96-well microtiter tray adaptor,
centrifuge for 30 minutes at 1500–3000 ¥ g. (see “Spin Column
Purification” on page 27 to calculate relative centrifugal forces.)
IMPORTANT
Do not refrigerate during the centrifugation run.
IMPORTANT
Be sure that these speeds are within the
manufacturer’s recommendations for the centrifuge.
5
Immediately invert the tray on paper towels. Place the inverted tray
and paper towel in the centrifuge for 1 minute at 150 ¥ g to remove
the supernatant.
IMPORTANT
This centrifugation step is important to ensure
complete removal of dye degradation products.
6
Add 50 µL of 70% ethanol to the tubes. Centrifuge for 5 minutes at
1500–3000 ¥ g.
7
Immediately repeat step 5 above.
25
Precipitating If there are only a few samples or you do not have access to a
Sequencing centrifuge with a 96-well tray adapter, then a single tube ethanol
Reactions in Single precipitation procedure may be used.
Tubes
Step
Action
1
Use a 1.5-mL Microcentrifuge tube for each sequencing reaction.
2
Prepare a fresh mix:
1.0 mL anhydrous ethanol (EtOH)
40 µL 3M sodium acetate (NaOAc) pH 4.6
3
To each 1.5-mL microcentrifuge tube add 25 µL of the
EtOH/NaOAc mix.
4
Pipet the entire contents of one extension reaction into one tube of
EtOH/NaOAc mix. Mix thoroughly by vortexing.
5
Spin the tubes in a microcentrifuge for 15–30 minutes at maximum
speed.
IMPORTANT
26
Do not refrigerate during the centrifugation run.
6
Carefully aspirate the supernatant with a pipette and discard.
7
Rinse the pellet with 50 µL of 70% ethanol.
8
Spin for 5 minutes in a microcentrifuge at maximum speed. Again,
carefully aspirate the supernatant and discard.
9
Dry the pellet in a vacuum centrifuge. Do not over-dry.
Spin Column We recommend Centri-Sep™ spin columns from Princeton Separations
Purification (P/N CS-901). This protocol is different from earlier spin column
protocols. The following changes have been made:
♦
Do not spin the column at speeds >735 ¥ g (see formula below).
♦
Do not spin more than 2 minutes.
♦
The rehydration time has been increased to 2 hours.
Tips for optimizing spin column purification:
♦
Use one column for each sample. Do not process more columns
than you can handle conveniently at one time.
♦
Load the sample in the center of the column bed without touching
the resin. Make sure that the sample does not touch the sides of the
column.
♦
Spin the column at 325–730 ¥ g for best results. Use the following
formula to calculate the best speed for your centrifuge:
g = 11.18 ¥
r ¥
(rpm/1000)2
where:
g = relative centrifugal force
rpm = revolutions per minute
r = radius of the rotor in cm
♦
Rehydrating the
Column
The entire procedure should be performed without interruption to
ensure optimal results. Do not allow the column to dry out.
Step
Action
1
Gently tap the column to cause the gel material to settle to the
bottom of the column.
2
Remove the upper end cap and add 0.8 mL of deionized water.
3
Replace the upper end cap and invert the column a few times to
mix the water and gel material.
4
Allow the gel to hydrate at room temperature for at least 2 hours.
Note
Rehydrated columns can be stored for a few days at
2–6 °C. Longer storage in water is not recommended. Allow
columns that have been stored at 2–6 °C to warm to room
temperature before use.
27
Step
5
Action
Remove any air bubbles by inverting or tapping the column and
allowing the gel to settle.
Using the Column Do not allow any interruptions after starting the steps below.
Step
1
Action
Remove the upper end cap first, then remove the bottom cap. Allow
the column to drain completely by gravity.
Note
If flow does not begin immediately, apply gentle pressure
to the column with a pipette bulb.
2
Insert the column into the wash tube provided.
3
Spin the column in a microcentrifuge at 730 ¥ g for 2 minutes to
remove the interstitial fluid.
4
Remove the column from the wash tube and insert it into a sample
collection tube (e.g., a 1.5-mL microcentrifuge tube).
5
Remove the extension reaction mixture from its tube and load it
carefully on top of the gel material.
6
Spin the column in a microcentrifuge at 730 ¥ g for 2 minutes.
Note
If using a centrifuge with a fixed-angle rotor, place the
column in the same orientation as it was in for the first spin. This is
important because the surface of the gel will be at an angle in the
column after the first spin.
7
Discard the column. The sample is in the sample collection tube.
8
Dry the sample in a vacuum centrifuge (no heat) for 10–15 minutes,
or until dry.
Do not over-dry.
28
Performing Codon 86 Group-Specific Sequencing
Overview In some heterozygote analyses, it is possible for multiple pairs of
different alleles to generate the same final sequence ladder. For
example, the heterozygous sequence from a DRB1*1102/1305
combination is identical to that from a DRB1*11041/1302 allele
combination. This situation is generically referred to as an ambiguous
heterozygote allele combination. In a majority of these cases, the allele
pairs are heterozygous at codon 86. Through the use of codon 86specific sequencing primers (P/N 4305023), the ambiguity can be
resolved.
The heterozygote PCR product generated in the low resolution SSPPCR is used as a sequencing template in two separate sequencing
reactions; one reaction uses a sequencing primer specific for the GTG
motif of codon 86 and the other reaction uses a sequencing primer
specific for the GGT motif. Homozygous sequences are generated in
each reaction, thus separating the two alleles, allowing definitive allele
assignment of each individual allele.
Performing Note Use MicroAmp Reaction Tubes or a MicroAmp Optical 96-Well
Sequencing Reaction Plate.
Reactions Note Ensure the thermal cycling is performed with the correct method and
the sample volume is set to 10 µL.
To perform the sequencing reactions:
Step
Action
1
Dilute PCR product 1:3 in DNA Diluent. For sufficient sample to
sequence with both codon 86 mixes, add 2 µL of PCR product to
6 µL of DNA Dilution Buffer.
2
To separate tubes/plate wells, add 8 µL of each of the codon 86specific Ready Reaction Mixes.
3
To each of the Ready Reaction Mixes in the tubes/plate wells, add
2 µL of diluted PCR product.
4
Seal the tubes with MicroAmp caps or cover the plate with a
MicroAmp 2400 or 9600 Full Plate Cover.
5
Centrifuge briefly in a benchtop centrifuge to combine the PCR
product and Ready Reaction Mix at the bottom of the tubes/plate
wells.
6
Place the tubes/plate in a thermal cycler.
29
To perform the sequencing reactions:
Step
7
8
9
(continued)
Action
Do 1 cycle of the following:
♦
Rapid thermal ramp to 96 °C
♦
96 °C for 10 seconds
Repeat the following for 20 cycles:
♦
96 °C for 10 seconds
♦
Rapid thermal ramp to 50 °C
♦
50 °C for 10 seconds
♦
Rapid thermal ramp to 60 °C
♦
60 °C for 2 minutes
Rapid thermal ramp to 4 °C and hold until ready to precipitate the
sequencing reactions.
To purify the samples, follow the ethanol precipitation protocol
described in “Precipitating Sequencing Reactions in a MicroAmp 9600
Tray” on page 25, or “Precipitating Sequencing Reactions in Single
Tubes” on page 26 or “Spin Column Purification” on page 27.
30
Recommendations for ABI PRISM 310 Genetic Analyzer Users
Overview If you have not previously used the dRhodamine-based sequencing
chemistries, the run module and dye set/primer (mobility) file will need
to be installed and the instrument (matrix) files made. The run module
and dye set/primer (mobility) file are provided on the HLA-DRB BDT
Supplemental Diskette. For more information on making the instrument
(matrix) files, see Appendix D on page 60.
Note
For more information on basic operation and safety, see the
ABI PRISM 310 Genetic Analyzer User’s Manual and the ABI PRISM 310 Genetic
Analyzer Site Preparation and Safety Guide.
Capillary To sequence on the ABI PRISM 310 instrument, use a 47-cm, green dot
Preparation capillary and Performance Optimized Polymer 6 (POP-6).
Preparing and IMPORTANT
To ensure adequate signal strength, perform the steps below
Loading Samples in the exact order they are given.
To prepare and load the samples from a MicroAmp 9600 tray:
Step
1
Action
Resuspend the DNA pellets in 15 µL of the recrystallized
formamide provided in the HLA Control and Loading Buffer Module.
! WARNING ! CHEMICAL HAZARD. Formamide is a
known teratogen. It can cause birth defects. Obtain a copy of
the MSDS from Applied Biosystems’ Web site or Fax-onDemand (see “Technical Support” on page 7). Wear
appropriate protective eyewear, clothing, and gloves. Wash
thoroughly after handling formamide.
IMPORTANT
Use only the recrystallized formamide supplied in
the HLA Control and Loading Buffer Module. Over time, formamide
decomposes to formate. Formate ions are injected preferentially
into the capillary, causing a loss of signal intensity.
2
Vortex the tubes, then centrifuge briefly.
3
Denature samples by placing the tray in a heating block or thermal
cycler for 2 minutes at 95±5 °C.
4
Vortex the tubes again, then centrifuge briefly.
5
Seal each tube securely using the strip septa.
6
Secure the strip septa with the retainer clips.
31
To prepare and load the samples from a MicroAmp 9600 tray:
Step
7
Action
Place the sample tray on the autosampler.
IMPORTANT
Do not let the denatured samples sit for more
than 48 hours before injection.
To prepare and load the samples in 0.5-mL Genetic Analyzer Sample
Tubes:
Step
1
Action
Resuspend the DNA pellets in 15 µL of the recrystallized
formamide provided in the HLA Control and Loading Buffer Module.
! WARNING ! CHEMICAL HAZARD. Formamide is a
known teratogen. It can cause birth defects. Obtain a copy of
the MSDS from Applied Biosystems’ webpage or Fax-onDemand (see p. 8). Wear appropriate protective eyewear,
clothing, and gloves. Wash thoroughly after handling
formamide.
IMPORTANT
Use only the recrystallized formamide supplied in
the HLA Control and Loading Buffer Module. Over time, formamide
decomposes to formate. Formate ions are injected preferentially
into the capillary, causing a loss of signal intensity.
2
Vortex the tubes, then centrifuge briefly.
3
Denature each sample by placing in a heating block or thermal
cycler for 2 minutes at 95±5 °C.
4
Vortex the tubes again, then centrifuge briefly.
5
Transfer the samples into 0.5-mL Genetic Analyzer Sample Tubes.
Label one tube for each sample.
IMPORTANT
Do not transfer to 0.5-mL tubes before the
sequences have been heat denatured. This may lead to lower or
inadequate signal strengths.
6
Seal each tube with a septum. Press the septa onto the tubes with
a slight twist of the thumb.
7
Place the tubes in the 48-well sample tray.
8
Place the sample tray on the autosampler.
IMPORTANT
Do not let the denatured samples sit for more
than 48 hours before injection.
32
Recommendations Enter sample information into the sample sheet.
for Sample
Verify that the Sample Names you enter for the forward and reverse
Naming
reactions are identical (except for sequence direction), as the
MatchTools software searches for sequence pairs with matching
Sample Names.
A useful format for Sample Name assignment is the specimen ID,
followed by the SSP-PCR group, a left-hand square bracket, and the
sequence direction (forward or reverse). For example, a sample that
amplified a DRB1 G3/11/6 might have the following file names:
♦
“Sample ID”.G3[F
♦
“Sample ID”.G3[R
IMPORTANT
Keep the Sample Name to 26 characters or less.
IMPORTANT
If you do not separate the sequence direction from the
Sample Name with the left-hand square bracket, the MatchTools software will
not correctly group the forward/reverse sequences of each SSP-PCR.
Setting up Default The ABI PRISM 310 Collection Program allows the user to set up default
Sample Names file names of each injection as preferences. Standardized names
attached to the sample name can be created and saved.
Step
Action
1
Open the ABI PRISM 310 Collection Software.
2
Under the Window menu, choose Preferences.
3
From Preferences, choose Default File Names.
4
For the Sample File, fill in the three fields as follows:
a.
5
Choose <tube number>, in the left pull-down window.
b.
Click in the middle field and type in <•> (option 8 key stroke).
c.
Choose <sample name> in the right pull-down window.
Click OK.
Sample Sheet The appropriate dye set/primer (mobility) file is “310 HLA-DRB BDT”
Menu choices supplied on the HLA-DRB BDT Supplemental Diskette.
Choose the correct instrument (matrix) file.
33
Filling in the Once the sample sheet is brought in to the Injection List, choose the run
Injection List module, “310 Run Module HLA-DRB BDT” for every injection. (This
module file is supplied on the HLA-DRB BDT Supplemental Diskette.
Place it in the Modules folder inside the 310 Collection Software folder.)
When this run module is chosen, the following information is displayed
in the injection list to the right of each sample:
Inj. Secs
Inj. kV
Run kV
Run °C
Run Time
30
1.0
15.0
50
28
Signal Strength For best results, the signal strengths of the analyzed data should range
from 100–1000 relative fluorescence units (RFU). Because of normal
variations among instruments and other factors, some adjustments may
be needed to modify the signal strength.
Increase or decrease the injection time and/or injection voltage
appropriately. Injection times greater than 60 seconds are not
recommended.
34
Performing Choose which data files to analyze, then analyze and print them using
Fluorescent Signal the Sequencing Analysis Software.
Analysis Setting the Stop Point in Analysis Parameters
Step
Action
1
Launch the Sequencing Analysis software and add data files to be
processed.
2
Open the data file and view the Raw Data.
3
Set the stop point to be at the end of the sequence run.
If too many bases are processed, the excess sequence will make it
difficult for the MatchTools software to properly define the sequence
orientation.
Sequence Analysis version 3.0 users may want to set the endpoint
in the Basecaller settings to 8 Ns in 10. Be aware that any
sequence region containing 8 Ns in 10 bases will be considered an
endpoint.
4
Return to the Sample Manager (Sample File Queue) window.
Highlight the name of the file just inspected and enter the
information for the Stop Point.
5
Enter information for other categories to be customized, such as
Basecaller (ABI-CE1), Peak 1 Location (Same as Start Point), Dye
Set/Primer file (310 HLA-DRB BDT), and Instrument file.
6
Repeat steps 1–3 for each sequence file.
7
Start analysis.
8
Review the newly analyzed sequences to ensure that the correct
analysis parameters were specified.
Note
If you encounter problems with the sequence data
generated, refer to the ABI PRISM DNA Sequencing Analysis
Software User’s Manual or the Troubleshooting Guide in
Appendix C on page 46.
35
Recommendations for ABI PRISM 377 DNA Sequencer Users
Overview If you have not previously used the dRhodamine-based sequencing
chemistries, the run module and dye set/primer (mobility) file will need
to be installed and the instrument (matrix) files made. The run module
and dye set/primer (mobility) file are provided on the HLA-DRB BDT
Supplemental Diskette. For more information on making the instrument
(matrix) files, see Appendix D on page 60.
Note
For more information on basic operation and safety, see the
ABI PRISM 377 DNA Sequencer User’s Manual and the ABI PRISM 377 DNA
Sequencer Safety Summary.
Gel Formulation Prepare a 36-cm well-to-read, 0.2 mm thick, 5% Long Ranger Gel. For
details, see the User Bulletin, “Improved Gel Formulations for Extended
Sequencing Read Lengths” (P/N 4303614).
! WARNING ! CHEMICAL HAZARD. Long Ranger Gel Solution
contains Acrylamide and/or Bis-Acrylamide. Acrylamide and BisAcrylamide are poisons, neurotoxins, irritants, carcinogens, and possible
teratogens. Acrylamide and Bis-Acrylamide sublime (the solid releases
toxic vapor) and are harmful if swallowed, inhaled, or absorbed through
the skin. Effects are cumulative. When handling, always wear personal
protective equipment (i.e., lab coat, safety glasses, and chemical resistant
gloves) and use in a well ventilated area. Thoroughly clean surfaces
subject to gel contamination (i.e., binder clips, combs, bench tops, etc.)
Preparing and IMPORTANT
To ensure adequate signal strength, perform the steps below
in
the
exact
order
they
are given.
Loading Samples
Preparing the Samples
Step
1
Action
Combine recrystallized formamide and blue dextran/EDTA solution
in a 5:1 ratio to prepare the sequencing loading buffer. Make fresh
for each use.
! WARNING ! CHEMICAL HAZARD. Formamide is a
known teratogen. It can cause birth defects. Obtain a copy of
the MSDS from Applied Biosystems’ Web site or Fax-onDemand (see p. 8). Wear appropriate protective eyewear,
clothing, and gloves. Wash thoroughly after handling
formamide.
36
Step
Action
2
Resuspend each DNA pellet in 4µL of sequencing loading buffer.
3
Vortex the tubes, then centrifuge briefly.
4
Denature each sample by placing in a heating block or thermal
cycler for 2 minutes at 95±5 °C.
5
Vortex the tubes again, then centrifuge briefly.
6
Place tubes on ice until ready to load.
Recommendations Enter sample information into the sample sheet.
for Sample
Verify that the Sample Names you enter for the forward and reverse
Naming
reactions are identical (except for sequence direction), as the
MatchTools software searches for sequence pairs with matching
Sample Names.
A useful format for Sample Name assignment is the specimen ID,
followed by the SSP-PCR group, a left-hand square bracket, and the
sequence direction (forward or reverse). For example, a sample that
amplified a DRB1 G3/11/6 might have the following file names:
♦
“Sample ID”.G3[F
♦
“Sample ID”.G3[R
IMPORTANT
Keep the Sample Name to 26 characters or less.
IMPORTANT
If you do not separate the sequence direction from the
Sample Name with the left-hand square bracket, the MatchMaker software will
not correctly group the forward/reverse sequences of each SSP-PCR.
Entering default The ABI PRISM 377 Collection program allows the user to set up default
file names for the file names as preferences. Standardized names attached to the sample
Sample File name can be created and saved.
Setting up default file names for a Sample File:
Step
Action
1
Open the ABI PRISM 377 Collection software.
2
Under the Window menu, choose Preferences.
3
Hold and release the mouse on Default File Names.
37
Setting up default file names for a Sample File:
Step
4
5
Action
For the Sample File, fill in the three fields as follows:
a.
Choose <lane number> in the left pull-down window.
b.
Click in the middle field and type in <•> (option 8 key stroke).
c.
Choose <sample name> in the right pull-down window.
Click OK.
Sample Sheet The appropriate dye set/primer (mobility) file is “377 HLA-DRB BDT”
Menu choices supplied on the HLA-DRB BDT Supplemental Diskette.
Choose the correct instrument (matrix) file.
Loading Sample Loading the samples
on the
Step
Action
ABI PRISM 377
1
Load the appropriate volume of each sample into a separate lane
as previously designated in the sample sheet:
♦
2 µL for a 24-lane gel
♦
1.5 µL for a 36-lane gel
IMPORTANT
lanes 1, 3, 5...
38
Load samples into alternate lanes, for example,
2
Prerun samples into the gel for 2 minutes.
3
Pause the electrophoresis prerun.
4
Load the remaining samples into alternate lanes, for example,
lanes 2, 4, 6...
ABI PRISM 377 Prior to collecting data, enter the settings for each of the
Run Parameters electrophoresis parameters: Comb, Matrix, Run Module (377 Run
Module HLA-DRB BDT), Well-to-Read Distance (36 cm), and Sample
Sheet.
The run parameters in the 377 HLA-DRB BDT run module are:
♦
Voltage: 1.5 kV
♦
Temperature: 51 °C
♦
Time: 4 hours
♦
Filter set E
Note
For information about general data collection, see the ABI PRISM 377
DNA Sequencer User’s Manual.
Performing Following Autoanalysis it may be necessary to process the data files.
Fluorescent Signal Choose which data files to analyze, then analyze and print them using
Analysis the Sequencing Analysis software.
Choosing Files for Analysis
Step
Action
1
Launch the Sequencing Analysis software. The Sample Manager
window (Sample File Queue in versions 2.1.1 and 2.1.2) appears.
2
Click the Add Files… button. A dialog box appears.
3
Double-click each file to be analyzed, then click Done.
4
Choose the ABI-100 basecaller for each file.
39
Generating DNA Sequences from Sample Files
Step
1
Action
Set the Start Point and Stop Point using the following procedure.
a.
Double-click on the first file to be analyzed and view the raw
data.
b.
Starting at the beginning of the raw data file, scroll along the
data by clicking and holding the right direction arrow at the
bottom of the dialog box.
c.
Continue scrolling until the cursor is beyond the broad
fluorescent peaks at the beginning of the sequencing ladder.
d.
Set the Start Point to the right of the last broad fluorescent
peak.
e.
Set the Stop Point to be at the end of the sequence run. If too
many bases are processed, the excess sequence will make it
difficult for the MatchMaker software to define the sequence
orientation.
Version 3.0 users may want to set the endpoint in the Basecaller
settings to 8 Ns in 10. Be aware that any sequence region
containing 8 Ns in 10 bases will be considered an endpoint.
2
Return to the Sample Manager (Sample File Queue) window.
Highlight the name of the file just inspected and enter the
information for the Start Point and Stop Point.
3
Enter information for other categories to be customized, such as
Peak 1 Location (Same as Start Point), Dye Set/Primer file (377
HLA-DRB BDT), and Instrument file.
4
Repeat steps 1–3 for each sequence file.
5
Start analysis.
6
Review the newly analyzed sequences to ensure that the correct
analysis parameters were specified.
Note
If you encounter problems with the sequence data
generated, refer to the ABI PRISM DNA Sequencing Analysis
Software User’s Manual or the Troubleshooting Guide in
Appendix C on page 46.
40
Assignment of HLA-DRB Allele Types to Sequence Files
Overview Each sequence data file is submitted to MatchTools Software for DRB1,
DRB3, DRB4, or DRB5 allele determinations. Refer to the MatchMaker
Allele Identification Software User’s Manual for more information.
Using MatchTools During a preliminary analysis, the MatchTools software performs the
Software for following functions:
Preliminary ♦ Pairs sequences having the same sample name.
Analysis
These are typically a forward and a reverse sequence from a single
PCR product
♦
Determines the orientation of each sequence within the pair
♦
Compares paired sequences with each other and generates a
consensus sequence
♦
Compares the consensus sequence against all of the alleles in the
library selected in the Batch Worksheet
MatchTools software then prepares a preliminary Batch Report listing
the:
♦
DRB allele type(s) that best fit(s) each consensus sequence
♦
Basecalls that differ within each pair of sequences
♦
Basecalls that display mismatches at constant positions or
polymorphic positions for all alleles in the library
♦
Any insertions or deletions
Note
At the 3´ end of the sequences, the reverse direction does not extend
as far as the forward sequence. The forward sequence continues to position
270 whereas the reverse sequence is compared against the library only to
position 262.
41
HLA-DRB Allele HLA-DRB Allele Libraries to use for each low resolution amplification
Libraries group/gene:
Allele Groups
Library Name
Codons Included
DR1, DR2, DR3/11/6, DR4,
DR8/12
DRB1.L155
14–90
DR7, DR9
DR79.L2
14–78
DR10
DR10.L1
30–88
DRB3, DRB5
DRB35.L17
14–90
DRB4
DRB4.L3
28–88
Using For manual editing of the sequence data, use MTNavigator Software to:
MTNavigator ♦ Import the Batch Worksheet containing all data files associated with
Software for
a given sample.
Editing
♦
–
The files are typically imported as a Forward/Reverse pair for
each SSP allele group or gene sequenced.
–
The sequences are offset such that the nucleotide numbering
corresponds to the cDNA sequences as published in the
nomenclature reports (i.e. the first position is nucleotide #40 in
the cDNA sequence). The first 39 bases are either in exon 1 or
from the first hypervariable region of exon 2 and therefore not
part of the allele library.
Generate shadow sequences for each pair of sequences to simplify
the comparison of basecalling differences between each pair of
sequences.
–
42
Basecall differences between the forward and reverse
sequences can be due to a variety of sources, including
mobility anomalies or base incorporation anomalies for
heterozygote positions (i.e., one base in a heterozygote pair
may not be detected at a sufficient level to be identified using
the threshold value set in the Batch Worksheet). See Table 3 on
page 49 for a list of all currently known reproducible anomalies.
♦
Perform manual editing of the sequences to resolve basecalling
differences between the two orientations.
When the editing is complete, save the changes to the sample files (use
the “Save to files...” command in the “Sequences” drop down menu).
Using MatchTools During a final analysis, use the MatchTools software to:
Software for Final ♦ Return to the preliminary analysis Batch Worksheet.
Analysis
♦
Generate a Final Batch Report by submitting the Batch Worksheet
to the MatchTools software. This report includes the:
–
HLA-DRB allele types that best match the edited sequences
–
Basecalls that display mismatches at constant positions or
polymorphic positions for all alleles in the library.
43
Appendix A. HLA-DRB Typing Results Template
Sample Form Use a form like the template below to indicate positive and negative
low-resolution grouping results for each sample.
Sample
Name
DRB1
G1
DRB1
G2
DRB1
G3/11/6
DRB1
G4
PCR Information
Thermal Cycler:
Date of Gel:
Operator:
Sequencing Cross-reference Information
Sequencer:
Date:
Folder Name:
Comments:
44
DRB1
G7
DRB1
G8/12
DRB1
G9
DRB1
G10
DRB3
Attach Photograph of Agarose Gel
DRB4
DRB5
DRB*
ALL
Appendix B. HLA-DRB Comparison Table
Low Resolution The following table presents the normal low resolution SSP-PCR
SSP-PCR Results expected results. These relationships are based on the normal
haplotype linkage patterns between DRB1 and the other three
expressed DRB genes.
DRB1
DRB1
DRB3
DRB4
DRB5
DRB1
DRB1
DRB3
DRB4
DRB5
1
–
–
–
3/11/6
4
+
+
–
2
–
–
+
3/11/6
7
+
+
–
3/11/6
+
–
–
3/11/6
8/12 (8)
+
–
–
4
–
+
–
3/11/6
8/12 (12)
+
–
–
7
–
+
–
3/11/6
9
+
+
–
8/12 (8)
–
–
–
3/11/6
10
+
–
–
8/12 (12)
+
–
–
4
7
–
+
–
9
–
+
–
4
8/12 (8)
–
+
–
10
–
–
–
4
8/12 (12)
+
+
–
1
2
–
–
+
4
9
–
+
–
1
3/11/6
+
–
–
4
10
–
+
–
1
4
–
+
–
7
8/12 (8)
–
+
–
1
7
–
+
–
7
8/12 (12)
+
+
–
1
8/12 (8)
–
–
–
7
9
–
+
–
1
8/12 (12)
+
–
–
7
10
–
+
–
1
9
–
+
–
8/12 (8) 8/12 (12)
+
–
–
1
10
–
–
–
8/12 (8)
9
–
+
–
2
3/11/6
+
–
+
8/12 (8)
10
–
–
–
2
4
–
+
+
8/12 (12)
9
+
+
–
2
7
–
+
+
8/12 (12)
10
–
–
–
2
8/12 (8)
–
–
+
9
10
–
+
–
2
8/12
(12)
+
–
+
2
9
–
+
+
2
10
–
–
+
45
Appendix C. Troubleshooting Guide
Overview The protocol and the HLA-DRB BDT Sequencing-Based Typing Kit will
provide data that allow high resolution allele assignments. However,
certain problematic situations may be encountered as detailed in the
tables below. Included in each table are descriptions of the problem,
possible causes, and suggestions for resolving the problem.
PCR
Symptom
Possible Cause
Action
No PCR
product or
weak product
Inappropriate cycling
parameters
Check the protocol and confirm
the correct method was used.
DNA not quantitated
correctly
Re-quantitate DNA and adjust to
10 ng/µL.
Degraded DNA
Evaluate on agarose gel and reextract the DNA if necessary.
PCR inhibitors in the
genomic DNA
Re-extract genomic DNA using
one of the recommended
methods (see “Blood Collection
and DNA Sample Preparation”
on page 14).
Symptom
Possible Cause
Action
Weak signal
strengths
Inefficient
precipitation of
sequence ladder
Re-sequence and precipitate,
referring to details in “Purifying
Sequencing Reactions” on
page 24.
Sequence ladder not
denatured properly
Re-sequence, precipitate, and
denature, referring to details in
“Preparing and Loading
Samples” on page 31 for the
ABI PRISM 310 DNA Sequencer
or “Preparing and Loading
Samples” on page 36 for the
ABI PRISM 377 DNA Sequencer.
Source of ethanol
Check reagent quality. Confirm
that the ethanol is absolute or
anhydrous.
Sequencing
46
Noisy baseline
Weak signal (less
than 50 fluorescent
units)
See “Weak signal strengths”
details above.
Signal too strong
Dilute the sequence ladder and
re-load.
Note
If the signal
is too strong,
multicomponent
analysis will not be
performed properly.
Broad
fluorescent
terminator
artifacts (dye
blobs)
For the ABI PRISM 310 user,
decrease injection time.
Incorrect run
module/filter set
Repeat the run using the correct
module (Run Module HLA-DRB
BDT).
Poor matrix
Make a new matrix. See
Appendix D on page 60.
Failure to perform
70% ethanol wash
Re-sequence and precipitate,
referring to details in “Purifying
Sequencing Reactions” on
page 24.
Failure to completely
remove supernatant
following anhydrous
ethanol precipitation
Re-sequence and precipitate,
referring to details in
“Precipitating Sequencing
Reactions in a MicroAmp 9600
Tray” on page 25 or “Precipitating
Sequencing Reactions in Single
Tubes” on page 26.
Note
For more general sequencing problems (e.g., poor resolution, base
spacing, etc.), refer to the ABI PRISM 310 Genetic Analyzer User’s Manual or
the ABI PRISM 377 DNA Sequencer User’s Manual.
47
MatchTools
Software
48
Symptom
Possible Cause
Action
Samples not
grouped
Sample names do not
match
Use the “Group these files”
command in the MatchTools
Worksheet menu.
Reverse
sequence not
automatically
reverse
complemented
by MatchTools
software
Too much sequence
beyond end of PCR
product
Re-analyze with Sequence
Analysis software; process only
to the end of the sequence.
Heterozygotes
not identified
by MatchTools
software
Base incorporation
anomaly
See known anomalies in Table 3
on page 49. Manually edit in
MTNavigator software and resubmit.
Allele type not
assigned to
good quality
data
Incorrect allele library
selected
Refer to “HLA-DRB Allele
Libraries” on page 42.
Anomalies Table Based upon sequences generated with this kit, reproducible anomalies
have been observed at some base positions. The table below
summarizes and gives examples of those anomalies that have been
encountered and may be useful for interpretation/editing of the
sequencing data. The majority of anomalies occur in only one
sequencing orientation for each base position and can be resolved by
reviewing data from the other orientation.
Table 3.
Anomalies
Sequence
Position Codon Orientationa
47
16
Forward
Heterozygote
Combinationb
1st Allele
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
All (constant
position)
CA
CM
mobility shift
All (constant
position)
CA
MM
mobility shift
Forward
100, 101
34
Forward
Forward
49
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
Heterozygote
Combinationb
1st Allele
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
112, 113
38
Forward
1301
1401
SY
GY
low C
incorporation
112, 113
38
Reverse
Complement
1301
1401
SY
GY
low C
incorporation
G
R
extra A (shoulder
from following A)
Forward
Reverse
136
46
All (constant
Reverse
Complement
position)
Reverse
50
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
155
52
Heterozygote
Combinationb
1st Allele
Reverse
All (constant
Complement
position)
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
G
R
extra A (shoulder
from following A)
G
S
low G with high
C background
Reverse
162
54
Reverse
All (constant
Complement
position)
Reverse
51
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
1st Allele
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
169, 170
57
Forward
1302
1303
RA
RR
low G
incorporation at
position 170
169, 170
57
Reverse
Complement
1302
1303
AR
RR
low G
incorporation at
position 169
Forward
Reverse
52
Heterozygote
Combinationb
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
170, 171
170, 171
57
57
Forward
Reverse
Complement
Heterozygote
Combinationb
1st Allele
2nd Allele
DRB3*
DRB3*
0101
0202
DRB3*
DRB3*
0101
0202
Correct Initial Base
Base Call
Call
Nature of
Anomaly
WY
WT
low C at position
171
WY
AY
low C
incorporation at
position 170
Forward
Reverse
53
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
201
67
Forward
Heterozygote
Combinationb
1st Allele
2nd Allele
All (constant
position)
Correct Initial Base
Base Call
Call
Nature of
Anomaly
C
Y
extra T (shoulder
from following T)
Forward
207-212
69-71
Forward
1301
1401
RSA SRR
RSA CAA
low G at
positions 210,
211, 212
207-212
69-71
Reverse
Complement
1301
1401
RSA SRR
RCA CAA
low G at
positions 208,
210, 211, 212
Forward
Reverse
54
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
212
71
Forward
Heterozygote
Combinationb
1st Allele
2nd Allele
1301
1303
Correct Initial Base
Base Call
Call
Nature of
Anomaly
R
A
low G at position
212
G
K
low G with T
background
noise
Forward
217
73
Forward
0301, or
DRB3 alleles
Forward
55
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
220
74
Reverse
Complement
Heterozygote
Combinationb
1st Allele
2nd Allele
0403
Correct Initial Base
Base Call
Call
Nature of
Anomaly
G
R
extra A (shoulder
from following A)
Y
T
low C
incorporation
Reverse
221
74
Forward (377)
Reverse
Complement
(310)
Forward
56
0801
1201
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
Heterozygote
Combinationb
1st Allele
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
256
86
Reverse
Complement
codon 86
heterozygotes
T
K
anomalous T
mobility (see
figure for codon
86 below)
257, 258
86
Reverse
Complement
codon 86
heterozygotes
KK
KT
incomplete
resolution of G
peaks
Forward
57
Table 3.
Anomalies
(continued)
Sequence
Position Codon Orientationa
261
87
Heterozygote
Combinationb
1st Allele
Reverse
All (constant
Complement
position)
2nd Allele
Correct Initial Base
Base Call
Call
Nature of
Anomaly
G
R
excess A signal
C
Y
extra T (shoulder
from previous T)
Reverse
267
89
Forward
All (constant
position)
Reverse
a. In this table, Reverse Complement refers to the reverse sequences after having been processed by MatchTools.
The bases described are those that will be observed in MTNavigator during the editing process.
b. Due to the large number of possible heterozygote allele combinations, the alleles listed in this table are for reference
only. For any given sample, it may be helpful to review the published allele sequences to determine whether the same
motif combinations are present in the sequences being reviewed.
58
IUB Codes The code:
A = Adenosine
R = A or G (puRine)
C = Cytidine
Y = C or T (pYrimidine)
G = Guanosine
K = G or T (Keto)
T = Thymidine
M = A or C (aMino)
B = C,G or T
S = G or C (Strong-3H bond)
D = A,G or T
W = A or T (Weak-2H bond)
H = A,C or T
N = aNy base
V = A,C or G
59
Appendix D. Instrument (Matrix) Files
Multicomponent Multicomponent analysis is the process that separates the four different
Analysis fluorescent dye colors into distinct spectral components. The four dyes
used in the ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready
Reaction Kit are dR110, dR6G, dTAMRA, and dROX.
Each of these fluorescent dyes emits its maximum fluorescence at a
different wavelength. During data collection on the ABI PRISM 310 and
377 instruments, the collection software collects light intensities from
four specific areas on the CCD camera, each area corresponding to the
emission wavelength of a particular fluorescent dye. Each of these
areas on the CCD camera is referred to as a “virtual” filter, since no
physical filtering hardware (like band-pass glass filters) is used. The
information that specifies the appropriate virtual filter settings for a
particular set of fluorescent dyes is contained in each appropriate run
module.
In the dRhodamine dyes, dR110 emits at the shortest wavelength and
is detected as blue, followed by dR6G (green), dTAMRA (yellow) and
dROX (red). Although each of these dyes emits its maximum
fluorescence at a different wavelength, there is some overlap in the
emission spectra among the four dyes. The goal of multicomponent
analysis is to isolate the signal from each dye so that there is as little
noise in the data as possible.
The precise spectral overlap between the four dyes is measured by
running DNA fragments labeled with each of the dyes in separate lanes
of a gel or in separate injections on a capillary. These dye-labeled DNA
fragments are called matrix standard samples.
The Data Utility software then analyzes the data from each of these four
samples and creates an instrument (matrix) file. The instrument file
contains tables of numbers with four columns and four rows. These
numbers are normalized fluorescence intensities and represent a
mathematical description of the spectral overlap that is observed
between the four dyes.
Multicomponent analysis of sequencing data is performed automatically
by the Sequencing Analysis software, which applies a mathematical
matrix calculation (using the values in the instrument file) to all sample
data.
60
Making a Matrix An instrument file can be made from matrix standards or it can be made
from a Sample File from a sample file. Using a sample file requires fewer steps than
running matrix standards, however the matrix made from a sample file
may be different from one made from matrix standards. The quality of a
matrix file made from a sample file depends on the quality of the sample
file used.
This section describes making a matrix using a BigDye Terminator
sequencing standard or a BigDye Terminator sequencing data file. Such
an instrument file, containing all three matrices, can be used with either
dRhodamine or BigDye chemistries.
For more information on creating a matrix using matrix standards, refer
to the ABI PRISM dRhodamine Matrix Standards User Bulletin (P/N
904917).
If problems arise, such as spectral overlap in the analyzed data, a new
matrix can be made from any terminator sequencing sample by
following the instructions below.
Run the Make one sample of the BigDye Terminator Cycle Sequencing
Sequencing Standard (P/N 4304154) following the instructions on the product insert.
Standard If using a sequencing data file, follow the steps below substituting the
appropriate file name.
Make Copies of the Make three duplicate copies of the BigDye terminator standard run
Standard Run before creating a matrix:
Step
Action
1
Open the Run folder, and highlight the sample file by clicking on
the name.
2
Open the File menu and choose Duplicate. A duplicate is
created, called “Sample file copy”.
3
Open the File menu and choose Duplicate two more times to
create “Sample file copy-2” and “Sample file copy-3.”
61
View Matrix Run Use the Data Utility software to view raw data of the matrix runs:
Raw Data
Step
Action
1
Launch the Data Utility software.
2
Under File, choose Open.
3
Search for the Run Folder containing the four sample files (one
original and 3 duplicates).
4
Click on the first sample. The corresponding open windows are:
♦
Raw data
♦
Analyzed
♦
EPT
♦
Information
5
Verify that the baseline is stable and peak heights are above 100
fluorescent units in the raw data.
6
Choose a scan point (e.g. 2000) that occurs after the first peaks,
though still in the beginning of the run.
.
Generate the The matrix standards are analyzed mathematically to create matrices.
Terminator Matrix
To generate a matrix :
Step
Action
1
From the Utilities menu, select Make Matrix... The Make Matrix
dialog box appears.
2
Designate the files for C, A, G, and T data.
Example:
62
a.
Click the C button, then choose “Sample file” in the Run
Folder.
b.
Do the same for the A (“Sample file copy”), G (Sample file
copy-2”), and T (“Sample file copy-3”) buttons.
c.
Use the default settings of 2000 for Start at and 1500 for
Points.
3
Click New File, name the matrix appropriately (e.g., HLA BDT),
and save it to the ABI Folder in the System folder.
4
Click T7 Terminator Matrix.
5
Click OK. The message “Make matrix successfully completed”
appears.
To generate a matrix
Step
6
(continued):
Action
If an error message occurs, modify the default values in the Make
Matrix dialog box. Make a new matrix.
Note
If you get an error message, and the software will not make a matrix,
you may have designated the wrong files. It is possible, though rare, that the
signal is too weak to make a matrix. If the signal is inadequate to make a matrix,
rerun the standard or use another dRhodamine/BigDye chemistry sequencing
run.
Complete the Repeat steps 1–5 on page 62 for making the Dye Primer Matrix and the
Other Matrices Taq Terminator Matrix of the instrument file.These two other matrices
are needed by the sequencing analysis software to analyze a BigDye
Terminator sequencing sample.
Be sure to select Update File instead of New File when repeating
step 3. The end result is a complete instrument file that can be used for
analyzing Big Dye and dRhodamine chemistries.
63
Evaluate Matrix To evaluate the quality of the matrix:
Quality
Step
Action
1
Launch the Sequencing Analysis software.
2
In the Sample Manager, add the sample file used to make the
matrix.
3
Select the new matrix under Instrument File.
4
Reanalyze the data file.
5
Look at the Analyzed data window.
Peaks of only one color should be seen (Figure 1).
Peaks under peaks indicate a poor or incorrect matrix (Figure 2).
6
If the sequence quality is poor, do one of the following:
a.
Modify the Make Matrix default values.
b.
See Note on preceding page.
Figure 1 Correct matrix
Figure 2 Incorrect matrix
64
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Printed in the USA, 7/2001
Part Number 4305024 Rev. 2
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