Clsi MM9 A

MM9-A
Vol. 24 No. 40
Replaces MM9-P
Vol. 24 No. 18
Nucleic Acid Sequencing Methods in

Diagnostic Laboratory Medicine; Approved
Guideline
This document addresses automated, PCR-based, dideoxy-terminator, and primer

extension sequencing done on gel- or capillary-based sequencers. Topics covered include
specimen collection and handling; isolation of nucleic acid; amplification and sequencing
of nucleic acids; interpretation and reporting of results; and quality control/assessment
considerations as appropriate.
A guideline for global application developed through the NCCLS consensus process.
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MM9-A
ISBN 1-56238-558-5
Volume 24 Number 40 ISSN 0273-3099
Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;
Approved Guideline
Michael A. Zoccoli, Ph.D.
Maria Chan, Ph.D.
James C. Erker
Andrea Ferreira-Gonzalez, Ph.D.
Ira M. Lubin, Ph.D., FACMG
Abstract
Sequencing methods for genotyping have moved from the research laboratory into the clinical laboratory. Sequencing is an assay
format of choice for very high complexity genotyping, especially when hundreds or thousands of bases of genetic sequence are
analyzed. NCCLS document MM9-A—Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved
Guideline addresses automated, PCR-based, dideoxy-terminator, and primer extension sequencing done on gel- or capillary-
based sequencers. Topics covered include specimen collection and handling, isolation of nucleic acid, amplification and
sequencing of nucleic acids, and interpretation and reporting results. Quality control/assessment considerations are addressed for
each step of the process as appropriate.
NCCLS. Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline. NCCLS document MM9-A
(ISBN 1-56238-558-5). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
THE NCCLS consensus process, which is the mechanism for moving a document through two or more levels of review by the
healthcare community, is an ongoing process. Users should expect revised editions of any given document. Because rapid
changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace
outdated editions with the current editions of NCCLS documents. Current editions are listed in the NCCLS Catalog, which is
distributed to member organizations, and to nonmembers on request. If your organization is not a member and would like to
become one, and to request a copy of the NCCLS Catalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100;
Fax: 610.688.0700; E-Mail: exoffice@nccls.org; Website: www.nccls.org
Number 40 NCCLS
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system,
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Reproduced with permission, from NCCLS publication MM9-A—Nucleic Acid

Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline (ISBN 1-
56238-558-5). Copies of the current edition may be obtained from NCCLS, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the
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To request such permission, address inquiries to the Executive Vice President, NCCLS, 940 West Valley
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Copyright ©2004. The National Committee for Clinical Laboratory Standards.
Suggested Citation
(NCCLS. Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline.
NCCLS document MM9-A [ISBN 1-56238-558-5]. NCCLS, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2004.)
Proposed Guideline
June 2004
Approved Guideline
December 2004
ISBN 1-56238-558-5
ISSN 0273-3099
ii
Volume 24 MM9-A
Committee Membership
Area Committee on Molecular Methods

Russel K. Enns, Ph.D. Carolyn Sue Richards, Ph.D., Linda Ivor
Chairholder FACMG Gen-Probe Inc.
Cepheid Oregon Health Sciences University San Diego, California
Sunnyvale, California Portland, Oregon
Robert B. Jenkins, M.D., Ph.D.
Roberta M. Madej, M.S., M.T. Advisors Mayo Clinic
Vice-Chairholder Rochester, Minnesota
Roche Molecular Systems, Inc. Dale H. Altmiller, Ph.D.
Pleasanton, California O.U. Medical Center Alan L. Landay, Ph.D.
Edmond, Oklahoma Rush-Presby.-St. Lukes Medical
Larry E. Bockstahler, Ph.D. Center
FDA Center for Devices and Lee Ann Baxter-Lowe, Ph.D. Chicago, Illinois
Radiological Health University of California,
Rockville, Maryland San Francisco Richard S. Schifreen, Ph.D.,
San Francisco, California DABCC
Frederick S. Nolte, Ph.D. Promega Corporation
Emory University Hospital Mark Evans, Ph.D. Madison, Wisconsin
Atlanta, Georgia American Medical Association
Chicago, Illinois Janet L. Wood, M.T.(ASCP)
Timothy J. O’Leary, M.D., Ph.D. BD Diagnostic Systems
Armed Forces Institute of Pathology Sparks, Maryland
Silver Spring, Maryland
Subcommittee on Nucleic Acid Sequencing
Michael A. Zoccoli, Ph.D. Staff

Chairholder
Celera Diagnostics Lois M. Schmidt, D.A.
Alameda, California Staff Liaison
NCCLS
Maria Chan, Ph.D. Wayne, Pennsylvania
FDA Center for Devices and
Radiological Health Donna M. Wilhelm
Rockville, Maryland Editor
NCCLS
James C. Erker Wayne, Pennsylvania
Abbott Laboratories
Abbott Park, Illinois Melissa A. Lewis
Assistant Editor
Andrea Ferreira-Gonzalez, Ph.D. NCCLS
Medical College of Virginia Wayne, Pennsylvania
Richmond, Virginia
Ira M. Lubin, Ph.D., FACMG

Geneticist
Centers for Disease Control and
Prevention
Atlanta, Georgia
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Volume 24 MM9-A
Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope..........................................................................................................................................1
2 Introduction................................................................................................................................1
3 Standard Precautions..................................................................................................................1
4 Terminology...............................................................................................................................2
4.1 Definitions ....................................................................................................................2
4.2 Abbreviations and Acronyms .......................................................................................4
5 Specimen Handling....................................................................................................................5
5.1 Specimen Collection, Transport, and Storage...............................................................5
5.2 Specimen and Nucleic Acid Retention .........................................................................6
5.3 Specimen Quality Assurance ........................................................................................6
6 Isolation of Nucleic Acid ...........................................................................................................6
6.1 Extraction of Nucleic Acid ...........................................................................................6
7 Amplification .............................................................................................................................8
7.1 Primer Design and Synthesis for PCR and Sequencing................................................8
7.2 Amplification Parameters .............................................................................................9
7.3 Practices to Aid in Contamination Control .................................................................10
7.4 Quality of Sequencing Template ................................................................................11
7.5 PCR Product Purification............................................................................................11
7.6 Quantity and Quality of DNA Template.....................................................................13
8 Sequencing...............................................................................................................................13
8.1 Sequencing Reactions .................................................................................................13
8.2 Controls and Reference Standards ..............................................................................15
8.3 Assay Verification ......................................................................................................15
8.4 Instrument Setup .........................................................................................................16
8.5 Electrophoresis............................................................................................................17
8.6 Data Examination .......................................................................................................17
9 Data Analysis ...........................................................................................................................19
9.1 Sequence Review........................................................................................................19
9.2 Clinical Interpretation .................................................................................................22
9.3 Reports ........................................................................................................................24
References.............................................................................................................................................26
Summary of Consensus/Delegate Comments and Committee Responses............................................28
The Quality System Approach..............................................................................................................30
Related NCCLS Publications................................................................................................................31

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Volume 24 MM9-A
Foreword
Nucleic acid sequencing is evolving rapidly as a diagnostic technique in the clinical laboratory.
Applications of this analytic approach are seen in several areas, including cancer testing (genetic testing
for cancer predisposition, assessment of gene mutations in cancer), genetics (carrier testing and diagnosis
of genetically transmitted diseases), and microbiology (viral genotyping, sequences associated with drug
resistance). Sequencing is an assay format of choice for very high complexity genotyping, especially
when hundreds or thousands of bases of genetic sequence are analyzed. Recently, sequencing-based tests
for HIV genotyping for assessing drug resistance have received FDA clearance for in vitro diagnostics
use. Currently, some laboratories utilize manual or automated assays developed in-house (“home-brew”),
while others use commercially available reagents and kits. There is a need for standardization of many
aspects of the sequencing process. While the chemistry of the reactions may be driven by a particular
supplier, sample handling, nucleic acid preparation and assessment, nucleic acid amplification,
sequencing and sequence data assessment, and reporting of results are independent of any kit that might
be used. This guideline is limited to automated, PCR, dideoxy-terminator, and primer extension
sequencing done on gel- or capillary-based sequencers.
Obtaining accurate clinical results from sequencing-based methods requires control of the process from
the acquisition of the sample to the interpretation of the sequence obtained. With the increase in the use
of laboratory-developed (“home-brew”) sequencing-based genotyping assays and commercially available
sequencing-based genotyping kits, a guideline for the development, verification, validation, and
implementation of sequencing-based assays is required to guide laboratories and manufacturers. This
guideline provides recommendations for all aspects of the sequencing process including specimen
collection and handling, isolation of nucleic acid, amplification and sequencing of nucleic acids, and
general interpretation and reporting of genotyping results.
It is hoped that this guideline will serve as a roadmap for laboratories to use in guiding themselves in
implementing sequencing-based genotypic testing.
Key Words
Capillary electrophoresis, dideoxy-terminators, electrophoresis, gel electrophoresis, nucleic acid, primer,

sequencing
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Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;

Approved Guideline
1 Scope
This guideline specifies recommendations for all aspects of the sequencing process including specimen
collection and handling, isolation of nucleic acid, amplification and sequencing of nucleic acids, and
general interpretation and reporting of genotyping results. It is the intent of this document to provide
instruction for verifying that the sequence obtained is accurate and suitable for subsequent interpretation;
to address general interpretation of the sequence; and to provide quality control/assessment considerations
for each step of the process as appropriate.
The intended users of this guideline are manufacturers and laboratories involved in the development,
verification, validation, and implementation of sequencing-based assays.
This guideline:
• does not address emerging methodologies such as array-based sequencing but is limited to automated,
PCR, dideoxy-terminator, and primer extension sequencing done on gel- or capillary-based
sequencers; and
• is not intended to provide very specific guidelines for interpreting every possible medical
consequence that can be discerned from analyzing individual sequencing results.
2 Introduction
With the sequencing of the human genome and an increasing number of other organisms, sequencing is
fast becoming an important tool for genotyping in molecular diagnostics. Sequencing is routinely used in
genotyping infectious disease organisms such as HIV and HCV. Additionally, in tissue typing for
transplantation, high resolution HLA typing is done by sequencing. Emerging applications for use of
sequencing in cancer biology, genetic susceptibility to disease, and understanding of complex genetic
diseases are coming into practice. Genotypic testing has already demonstrated clinical utility in patient
management and has resulted in genotyping moving rapidly out of the research laboratory and into the
clinical laboratory setting. Sequencing is an assay format of choice for very high-complexity genotyping,
especially when hundreds or thousands of bases of genetic sequence are analyzed. With the increase in
the use of laboratory-developed (“home-brew”) sequencing-based genotyping assays and commercially
available sequencing-based genotyping kits, a guideline for the development, verification, validation, and
implementation of sequencing-based assays is required to guide laboratories and manufacturers. It is
hoped that this guideline will serve as a roadmap for laboratories to use in guiding themselves in
implementing sequencing-based genotypic testing.
3 Standard Precautions
Because it is often impossible to know what might be infectious, all patient and laboratory specimens are
treated as infectious and handled according to “standard precautions.” Standard precautions are guidelines
that combine the major features of “universal precautions and body substance isolation” practices.
Standard precautions cover the transmission of all infectious agents and thus are more comprehensive
than universal precautions which are intended to apply only to transmission of blood-borne pathogens.
Standard and universal precaution guidelines are available from the U.S. Centers for Disease Control and
Prevention (Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital
Epidemiology. CDC. 1996;17(1):53-80 and MMWR 1988;37:377-388). For specific precautions for
An NCCLS global consensus guideline. ©NCCLS. All rights reserved. 1
Number 40 NCCLS
preventing the laboratory transmission of all infectious agents from laboratory instruments and materials
and for recommendations for the management of exposure to all infectious disease, refer to the most
current edition of NCCLS document M29—Protection of Laboratory Workers from Occupationally
Acquired Infections.
4 Terminology
4.1 Definitions
Accuracy (of measurement) – Closeness of the agreement between the result of a measurement and a
true value of the measurand (VIM93)1; See the definition of Measurand, below.
All base accuracy – Calculated by determining the percentage of the bases called that agree with the
expected base call in the reference sequence.
Allele – In Genetics, any of several forms of a gene that is responsible for hereditary variation; NOTE: A
pseudoallele is an almost-identical sequence to the allele found elsewhere in the genome.
Amplification – The enzymatic replication in vitro of a target nucleic acid; NOTE: The polymerase
chain reaction (PCR) is a commonly used method of amplification.
Annealing – The hybridization of two complementary strands of nucleic acid, as in the hybridization of a
probe or primer with the target DNA.
Antisense strand – Strand of DNA complementary to the sense strand.
Base call – Ability to distinguish presence of an adenosine (A), a thymidine (T), a cytidine (C), or a
guanosine (G) at a given position within a sequence ladder or compilation of overlapping sequence
ladders; NOTE: Positions may be ambiguous and represented by an S (G or C), W (A or T), K (G or T),
Y (C or T), M (A or C), R (A or G), B (C, G, or T), D (A, G, or T), H (A, C, or T), V (A, C, or G), or N
(any base).
Complementary – Describing the property of two strands of nucleic acid which can hybridize by
specific-base pairing between the nucleotides.
Compression – Artifacts that are produced during electrophoresis due to the formation of stable
secondary structures in the sequencing product DNA fragments; NOTES: a) The folded structures run
faster through the matrix than equivalent unfolded DNA fragments resulting in odd spacing, or bands very
close together followed by bands spaced further apart than normal; b) In some cases, bases can be lost
entirely.
Consensus sequence – The final sequence generated from a compilation of overlapping sequence ladders
following the completion of base calling at all positions; NOTE: The consensus sequence is believed to
be representative of the source nucleic acid.
Denaturation – The conversion of double-stranded DNA or RNA to a single-stranded state with minimal
secondary structure; NOTES: a) This is done by heating, increasing pH, or adding agents such as
formamide or urea; b) Once denatured, nucleic acid molecules are available for hybridization with a
primer or probe.
Diagnostic sensitivity – The proportion of patients with a well-defined clinical disorder whose test values
are positive or exceed a defined decision limit (i.e., a positive result and identification of the patients who
have a disease); NOTES: a) The clinical disorder must be defined by criteria independent of the test
2 An NCCLS global consensus guideline. ©NCCLS. All rights reserved.

Volume 24 MM9-A
under consideration; b) The term Diagnostic sensitivity (Europe) is equivalent to Clinical sensitivity
(U.S.).
Diagnostic/confirmatory testing – 1) Testing generally performed to evaluate the genetic status of

individuals at increased risk for a particular disorder due to a positive family history or symptoms; 2) A
test that confirms the presence or absence of a substance by another methodology or procedure that is
either more sensitive, more specific, or both; 3) A clinical condition as a follow up to testing previously
performed that indicated the patient being at higher risk for having a clinical condition.
Electropherogram – The visualization of a sequence ladder characterizing the signal strength, noise,
base spacing, and base calls as seen by the sequencing software; NOTE: This chart can be used as a tool
to evaluate the quality and accuracy of the sequence.
Electrophoresis – see Gel electrophoresis.
Extension/Elongation – The 5′ to 3′ synthesis of DNA starting from an annealed primer to generate a

complementary strand of DNA.
Gel electrophoresis – Separation of molecules in an electric field within a matrix of agarose or

polyacrylamide according to size and charge.
Genotype – 1) The genetic makeup of an organism, or group of organisms, with reference to a single
trait, set of traits, or an entire complex of traits; 2) The specific allelic composition of a gene, or set of
genes, established at the DNA level.
Hybridization – Base pairing of complementary strands of nucleic acid by hydrogen bond formation, the
binding of probe to specific nucleic acid sequences or amplification products; NOTE: Hybridization can
be performed with both nucleic acid target and probe in solution, or with either one bound to a solid
support such as a microtiter plate or membrane.
Measurand – Particular quantity subject to measurement (VIM93)1; NOTE: This term and its definition
encompass all quantities, while the commonly used term “analyte” refers to a tangible entity subject to
measurement. For example, “substance” concentration is a quantity that may be related to a particular
analyte.
Mismatch – Hybridization of two DNA or RNA strands that are less than 100% complementary.
Mutation – An abnormal variation in DNA sequence which is found in association with, or which
reflects a predisposition to disease; NOTE: Mutations must be distinguished from polymorphisms—
DNA variants found in a population that neither harbors, nor is predisposed to disease.
Noise – Unwanted background signal which may adversely affect the ability to distinguish a positive
signal.
Oligonucleotide – A small polynucleotide generally synthesized on a machine; NOTE: Oligonucleotides

are used as amplification primers or as probes.
Polymerase chain reaction (PCR) – A common method of DNA amplification, utilizing pairs of
oligonucleotide primers as start sites for repetitive rounds of DNA polymerase-catalyzed replication and
alternating with denaturation in successive heating-cooling cycles.
Polymorphism – The occurrence together in a population of two or more alternative genotypes, each at a
frequency greater than that which could be maintained by recurrent mutation alone; NOTE: A locus is

Number 40 NCCLS
arbitrarily considered to be polymorphic if the rarer allele has a frequency of 0.01, so that the
heterozygote frequency is at least 0.02.
Positive predictive value – The likelihood that an individual with a positive test result has a particular
disease, or characteristic, that the test is designed to detect.
Primer – An oligonucleotide that hybridizes with one strand of a DNA or RNA template, providing a free
3′–OH end at which a polymerase starts the synthesis of a new, complementary strand.
Resolution – In DNA sequencing, the ability to clearly distinguish between adjacent positions; NOTE:
Resolution is influenced by the parameters of electrophoresis, sequence ladder peak height (signal
strength), and peak width.
Ribonuclease – An enzyme that catalyzes the breakdown of RNA molecules into smaller components.
Sense strand – The strand of a duplex DNA that has the same nucleotide sequence as mRNA except that
T substitutes in DNA for U in RNA; NOTE: The sense strand is also called the coding strand. The other
strand, which is the actual template for mRNA synthesis, is the anticoding or antisense strand.
Sensitivity – The change in response of a measuring system or instrument divided by the corresponding
change in the stimulus (modified from VIM93)1; NOTES: a) The sensitivity may depend on the value of
the stimulus (VIM93)1; b) The sensitivity depends on the imprecision of the measurements of the sample.
Sequence – The order of nucleotides (A, C, G, T, or U) in a strand of DNA or RNA.
Signal strength – In sequencing, the peak height associated with the termination of a sequence product
leading to the generation of a sequence ladder; NOTE: Signal strength allows for the identification of a
base above the noise.
Specificity – The ability of a measurement procedure to measure solely the measurand; NOTE:
Specificity has no numerical value in this context; See Measurand above.
Target sequence – Area of nucleic acid to be detected and sequenced.
Validation – Confirmation through the provision of objective evidence, that requirements for a specific
intended use or application have been fulfilled (ISO 9000)2; NOTES: a) WHO defines validation as the
action {or process} of proving that a procedure, process, system, equipment, or method used works as
expected and achieves the intended result (WHO-BS/95.1793)3; b) The components of validation are
quality control, proficiency testing, validation of employee competency, instrument calibration, and
correlation with clinical findings.
Verification – Confirmation through the provision of objective evidence that specified requirements have
been fulfilled (ISO 9000)2; NOTE: A one-time process completed to determine or confirm test
performance characteristics before the test system is used for patient testing.
4.2 Abbreviations and Acronyms
A adenine
C cytosine
cDNA complementary DNA
dATP deoxyadenosine 5′ triphosphate
dCTP deoxycytidine 5′ triphosphate
ddATP dideoxyadenosine 5′ triphosphate

Volume 24 MM9-A
ddCTP dideoxycytidine 5′ triphosphate

ddGTP dideoxyguanosine 5′ triphosphate
ddNTP dideoxyribonucleotide
ddTTP dideoxythymidine 5′ triphosphate
dGTP deoxyguanosine 5′ triphosphate
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
dNTPs deoxyribonucleoside triphosphates
dTTP deoxythymidine 5′ triphosphate
dUTP deoxyuridine 5′ triphosphate
ExoI/SAP exonuclease I/shrimp alkaline phosphatase
FDA Food and Drug Administration
G guanine
HCV Hepatitis C Virus
HIV Human Immunodeficiency Virus
HPLC high performance liquid chromatography
mtDNA mitochondrial DNA
PCR polymerase chain reaction
PBMCs peripheral blood mononuclear cell
RNA ribonucleic acid
RNAse ribonuclease
T thymine
U uracil
UNG uracil N-glycosylase
5 Specimen Handling
The specimen container should be clearly labeled with a unique patient identifier. A requisition form and
clinical and family history when applicable, should accompany every sample. It is recommended that
each laboratory have written criteria for acceptance and rejection of specimens. For inherited disease
testing, issues with regard to the need for informed consent are often raised. The decision as to whether
informed consent is required before testing is carried out should take into account any applicable federal,
state, or local requirements. NCCLS documents MM1—Molecular Diagnostic Methods for Genetic
Diseases and MM5—Nucleic Acid Amplification Assays for Molecular Hematopathology provide
detailed considerations for the selection of relevant criteria for the above items.
5.1 Specimen Collection, Transport, and Storage
From the safety standpoint, the collection and handling of specimens for transport and storage should
proceed with the same precautions taken for any potential infectious materials, following current blood-
borne pathogen, infectious disease, and appropriate regulatory guidelines (see Section 3).
Appropriate specimen collection, handling, and transport conditions are critical to ensure specimen
integrity. Each laboratory should have written criteria for specimen collection, transport, and storage.
Specimen transport and storage conditions will depend upon the nucleic acid of interest, either DNA or
RNA. NCCLS documents MM1—Molecular Diagnostic Methods for Genetic Diseases and MM5—
Nucleic Acid Amplification Assays for Molecular Hematopathology provide detailed information
regarding appropriate specimen transport and storage conditions for a variety of specimens that could be
used for sequencing purposes.

Number 40 NCCLS
5.2 Specimen and Nucleic Acid Retention
It is recommended that each laboratory have written criteria for specimen and nucleic acid retention. A
primary concern with specimen and nucleic acid retention involves ethical and legal considerations such
as informed consent and specimen reuse for the same and/or other testing. Some of these issues could be
raised and documented during the informed consent process. These issues are currently being evaluated
by different government agencies and professional societies, and recommendations can be expected.
Until these recommendations and/or regulations are established, each laboratory should determine its own
specimen and nucleic acid retention policy.
5.3 Specimen Quality Assurance
Samples should be maintained for an appropriate period of time to confirm sequencing results as required
by regulation or recommended by laboratory practice standards. Patient samples or nucleic acids isolated
can be used for quality control purposes once samples are anonymized, unless the person from which the
sample came requests that the specimen not be used for this purpose. Regulations may require further
measures such as informed consent from the patient prior to sample use for quality control purposes.
6 Isolation of Nucleic Acid

Numerous manual and several automated procedures exist for extraction of nucleic acids from clinical
specimens to be used for sequencing. Several commercial nucleic acid isolation kits are available that
may simplify and reduce the time of this procedure. Commercial sequencing kits may or may not include
an extraction procedure as part of the kit. A key factor for the extraction of nucleic acids that are to be
sequenced is that they should be as pure as possible so as to remove all inhibitors, competing nucleic
acids, and other substances that may interfere with the amplification and cycle-sequencing reactions.
Each laboratory should evaluate various DNA and/or RNA purification systems for yield efficiency,
consistency, and nucleic acid purity before incorporation as a standard procedure.
6.1 Extraction of Nucleic Acid
The quality of sequencing results is directly related to the quality of the nucleic acid template. The
template for sequencing should be free from protein, RNA, ethanol, salts, and any organic contaminants.
The methods that are commonly used for extraction of nucleic acids that are to be sequenced are the same
as those for amplification. The main categories include organic extraction and solid phase extraction
methods.
6.1.1 Organic DNA Extraction Method
Organic extraction is a classical technique that uses organic solvents to separate the DNA from protein
and other contaminants. Cells are lysed using a detergent, and then mixed with phenol, chloroform, and
isoamyl alcohol. The correct salt concentration and pH are critical to ensure that contaminants are
separated into the organic phase and that DNA remains in the aqueous phase. DNA is usually recovered
from the aqueous phase by alcohol precipitation.
6.1.2 High Salt Method
A modification of the above method involves the use of high salt, detergent, and isopropanol to isolate
and precipitate DNA from the solution. This method uses a detergent to release DNA and denature
proteins from cells. RNA can be removed if necessary using RNase A. Proteins are precipitated by using
a high salt solution that contains ammonium acetate, followed by isopropanol precipitation of DNA. The
DNA precipitates out of solution in isopropanol because some of the salt from the protein precipitation
solution will carry over into the alcohol and the salt and alcohol together will dehydrate the DNA,
Volume 24 MM9-A
bringing it out of solution. This system provides high purity DNA preparations without the need for
organic solvents and can accommodate significant volume of starting specimen.
6.1.3 Solid Phase Extraction Methods
Solid phase extraction methods have almost replaced the organic extraction methods. Compared to the
traditional liquid-liquid extraction, the major advantages are avoidance of dangerous organic solvents like
chloroform and phenol, significant time savings, and the ultrahigh purity of the nucleic acid product.
6.1.3.1 Silica Technology
With the silica-based membrane method, nucleic acids are selectively adsorbed to the silica at high
concentrations of chaotropic salt such as guanidine hydrochloride, guanidine isothiocyanate, sodium
iodide, and sodium perchlorate. The bound DNA is washed, and then eluted with a low-salt buffer or
with water. This system provides high purity DNA preparations with a choice of purification formats such
as spin columns, strips, or 96-well plates.
6.1.3.2 Glass Fiber Technology
Cells are lysed during a short incubation with proteinase K in the presence of a chaotropic salt (guanidine
HCl), which immediately inactivates all nucleases. Cellular nucleic acids bind selectively to glass fiber
fleece in a special centrifuge tube. The nucleic acids remain bound while a series of rapid “wash-and-
spin” steps remove contaminating small molecules. Finally, low-salt elution removes the nucleic acids
from the glass fiber fleece. The process does not require precipitation, organic solvent extractions, or
extensive handling of the nucleic acids.
6.1.3.3 Anion-Exchange Methods
Solid-phase anion-exchange chromatography is based on the interaction between the negatively charged
phosphates of the nucleic acid and positively charged surface molecules on the substrate. DNA binds to
the substrate under low-salt conditions; impurities such as RNA, cellular proteins, and metabolites are
washed away using medium-salt buffers; and then ultrapure DNA is eluted using a high-salt buffer. The
eluted DNA is recovered by alcohol precipitation. The matrix used is a special anion exchanger, packed
into polypropylene columns. The sample binding takes place under low-salt concentration conditions.
During the washing and elution steps, the salt concentration is increased stepwise. This method is suited
for the rapid and efficient purification of DNA and RNA as well. Impurities are removed quantitatively
because of their different binding behavior.
On occasion, it may be necessary to concentrate or enrich the specimen before it can be amplified for
sequencing. Concentration of starting material is achieved by ways such as filtration or centrifugation.
Enrichment can be achieved by binding to probes and/or antibodies.
6.1.4 RNA Extraction Procedures
Isolation of RNA is more challenging than isolation of DNA. A significant problem encountered during
RNA extraction is the degradation of RNA by RNAses. RNAses are very difficult to inactivate and even
resist boiling. In addition to their presence within the cells of the tissue being extracted, these enzymes
can be easily introduced during laboratory manipulation from a variety of sources including sneezing,
hair, and fingerprints. Extra care is required to ensure that all reagents, glassware, and plasticware are
RNAse free. A number of commercial preparations are available that either inhibit or destroy this
enzyme. The most commonly used procedure is treatment of water, nontemperature-sensitive reagents,
nonvolatile reagents, glassware, and plasticware with diethylpyrocarbonate (DEPC) followed by
autoclaving. Chaotropic agents such as guanidinium isothiocyanate are generally used in the purification

Number 40 NCCLS
of RNA. These agents are capable of dissolving cellular membranes, causing nucleoproteins to dissociate
from nucleic acid and inactivating ribonucleases. Purification of RNA can be achieved by organic or
nonorganic procedures. The organic and nonorganic procedures for isolating RNA are similar to the ones
already described for DNA with slight modifications. As a final step in purification, RNA should be
stored in a solution containing an appropriate amount of RNase inhibitors.
6.1.5 Automated Nucleic Acid Extraction
Different manufacturers have introduced several stand-alone robotic automatic nucleic acid purification
instruments with different success. A number of these automatic extractors use a modification of a manual
procedure such as high-salt, glass fiber, and silica-based methods. Manufacturers of automatic extractors
usually provide kits for DNA or RNA extraction from a variety of specimen sources. It is anticipated that
fully automated instruments will be developed and implemented in the near future.
7 Amplification
Sequencing of a particular nucleic acid target requires, first of all, adequate quantity of the target
sequence. In the majority of the cases, it is not possible to direct the sequence straight from the patient
specimens. PCR has proven invaluable because it allows the amplification of a specific nucleic acid
sequence without having to first clone in a microbial host.
There are two main elements that are crucial for successful sequencing of PCR products. Primer selection
and amplification conditions should be optimized to yield sufficient quantities of the specific target
without producing significant background. For additional information regarding amplification, refer to
NCCLS documents MM3—Molecular Diagnostic Methods for Infectious Diseases and MM5—Nucleic
Acid Amplification Assays for Molecular Hematopathology.
7.1 Primer Design and Synthesis for PCR and Sequencing
PCR uses repeated cycles of template denaturation, primer annealing, and polymerase extension to
amplify a specific target sequence. The oligonucleotide primers determine the specific target sequence
that is amplified.
Primers used for PCR must be designed using a known sequence for the target of interest. Sequencing
primers can be designed as well using a known sequence from the target, or it is possible to add several
noncomplimentary bases at the 5′-end of the PCR primer to generate a unique nontarget sequence for the
sequencing primer. Sequencing can also be done using the PCR primers but this is not advisable. Several
factors need to be taken into consideration when designing primers whether for PCR or sequencing. They
are as follows:
• Primers should be at least 18 to 28 nucleotides in length to ensure good hybridization.
• Match Tm/Td of the primers to each other.
• The Tm selected should be optimized for the maximum sensitivity and specificity of the PCR and
sequencing reactions.
• Avoid runs of an identical nucleotide, especially four or more runs of Gs.
• Keep C-G content between 40 to 70%, preferably 50 to 55%.
• Design primers that are not complementary to each other.

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• Avoid complementarity at the 3′ ends.
• Avoid complementarity at the 5′ ends.
• Avoid palindromic sequences, particularly at the 3′ end, as they can form secondary structures.
• Avoid all known SNPs by checking primers against SNPs databases.
• Sequence conservation should be taken into consideration during primer design. If possible, primers
should be designed in the most conserved region of the sequence of interest.
• Whenever possible and/or appropriate, primers should be designed at least 50 bases in the intron
sequences in order to be able to interrogate intron/exon sequences with quality sequence data.
• Ambiguous positions are allowed, but they should be avoided in the 3′-most positions.
• For sequencing, the primers should not be closer than 40 nucleotides to the sequence of interest due to
the difficulty of reading sequences immediately after the primer.
There are a number of different computer software programs available that can greatly assist with PCR
and sequencing primer selection and design. Some of these programs are freely available on the Internet
at: genome-www2.stanford.edu/cgi-bin/SGD/web-primer and frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.
There are also commercial sites on the Internet that provide access to computer software programs. It is
advisable to perform homology checks against known sequences to identify possible homology of the
primers with other known sequences that might impact primer specificity. Pseudoalleles or almost-
identical sequences found elsewhere in the genome could sometimes confound sequencing reactions and
interpretations. Sequence databases may reveal the presence of these, and efforts should be made for
selective amplification of the desired sequence. While these rules and software can aid in primer design,
practical optimization of amplification conditions to ensure sensitivity and specificity must be performed.
Sequence primer spacing is partially dependent upon the sequencing technology used. The optimal
spacing between the primers is defined by the sequencing instrument and technology employed. Newer
sequencing chemistries and methodologies will further increase the sequence length attainable. For many
formats in use today, primers should be placed every 200 to 600 bases on both the sense and antisense
strands for accurate base calls to be made.
The dyes for dye-labeled primers should be chosen to be compatible with the detection system of the
sequencing platform used. The dyes and linkages should be stable to the temperature cycling between 40
and 92 °C, which occurs in cycle sequencing. The fluorescent dyes used for sequencing are all photolabile
to some extent and should be protected from light during storage and use. Purified dye-labeled primers
are readily available from commercial sources and should be qualified by sequencing a control or
reference standard prior to use with clinical samples.
7.2 Amplification Parameters
While there are standard conditions that will amplify most target sequences, each new PCR application is
likely to require optimization. Some problems that are often encountered are: lack of PCR product, low
yield of the desired product, the presence of nonspecific background bands due to mispriming or
misextension of the primers, and formation of primer-dimer that competes for amplification with the
desired product.

Number 40 NCCLS
The following PCR parameters need to be taken into consideration and, if necessary, optimized:
• amount of starting template;
• primer concentration;
• type of enzyme and enzyme concentration;
• use of chemically modified thermostable DNA polymerases that provide automatic hot start, allowing
template DNA and primers to be mixed together and held at a temperature above the threshold of
nonspecific binding of primer to template before nucleic acid amplification occurs;
• magnesium ion concentration;
• buffer composition and pH;
• annealing temperature; and
• number of cycles.
Various additions or cosolvents such as dimethyl sulfoxide (DMSO), glycerol, and bovine serum albumin
have been shown to improve amplification in many applications. Careful evaluation of these parameters
will greatly increase the specificity of the amplification and decrease the amount of nonspecific products
that can interfere with the sequencing reaction.4-17
7.3 Practices to Aid in Contamination Control
Due to the ability of PCR to amplify from minute amounts of target template or already amplified product
(amplicon), laboratories are strongly encouraged to implement measures to avoid template and/or
amplicon contamination. This can best be accomplished by developing a workflow process to minimize
the potential for amplicon and target contamination.18-22 Physical separation of reagent preparation,
specimen processing and nucleic acid extraction, and processing of the amplified product should be
carefully designed. Unidirectional workflow must be strictly followed whereby personnel perform tasks
in the clean areas first, and only after completing their tasks there, move into the postamplification area.
In addition, supplies and instruments should be dedicated to each area. Protection of countertops with
absorbent plastic-backed paper must be changed after each use as paper can collect dust and with it,
amplified material. The use of bleach appears to be the most generally accepted method for
decontaminating surfaces. Decontamination of countertops, laboratory equipment, and furniture should
be performed by wiping with a freshly made 5% to 10% solution of bleach and then with a 75% ethanol
solution or even water to dilute the bleach that could corrode the surfaces. All pipette tips should contain
a hydrophobic filter in order to avoid any contamination of the tip of the pipettor with template or
amplified material. Moreover, these tips should be discarded into a container which can be completely
sealed before discarding them. Gloves should be changed periodically or as soon as any contamination is
suspected.
A chemical means of carry-over prevention is through the incorporation of deoxyuridine triphosphate

instead of deoxythymidine triphosphate into the amplicon during the amplification procedure. In this
procedure, deoxyuridine triphosphate is incorporated into every amplicon, which makes them susceptible
to destruction by uracil N-glycosylase (UNG) and heat. If uracil-containing amplicon is incorporated by
accident into another sample, one can eliminate this carry-over contamination by treating the sample prior
to amplification with UNG. Treatment with UNG will result in destruction of the uracil-containing
amplicon. The uracil-containing amplicon is the substrate for UNG, while the template is not. The
mechanism behind this chemical reaction is that UNG removes uracil residues from the amplicon and

Volume 24 MM9-A
leaves an intact phosphodiester backbone in the amplicon. During the first denaturation step of the
amplification procedure, the phosphodiester bond breaks at the sites were the uracil residues were located.
The fragment amplicon is no longer able to act as a template. Use of dUTP and UNG has been shown to
be effective to control contamination if the uracil-containing amplicon does not exceed the amount
between 106 and 107 copies per reaction. Uracil-containing amplicons can be sequenced with good
success under the same conditions used for the usual thymidine-containing amplicons.
One of the shortcomings of this procedure is the need to optimize the amount of dTTP and dUTP. The
dTTP and dUTP ratio is optimized to get a good yield of PCR product from the target. But the dUTP
level needs to be high enough to ensure that UNG treatment of the amplicon will eliminate its ability to be
amplified. Thus, it should be possible to incorporate this contamination control technology into almost all
sequencing assays.
7.4 Quality of Sequencing Template
Evaluation of substances that could interfere with the ability of amplification and sequencing must be
performed. Interfering substances are components present in the patient specimen that interfere with the
amplification of the specific target. There are mainly two sources of interfering substances. Exogenous
interfering substances are substances that are introduced to the specimen such as anticoagulant or
substances that are given to the patient that may affect the test ability to detect or measure the target of
interest. These could be drugs, parenteral nutrition, etc. On the other hand, endogenous interfering
substances (e.g., lipids, bilirubin) could be a result of pathological conditions.
DNA template quality and quantity is critical for good quality sequencing results. Poor template quality is
the most common cause of sequencing problems. Some of the problems derived from poor quality
template are noisy data, peaks under peaks, weak signal, and even no usable sequence data. A common
problem affecting template quality is the presence of contaminants such as proteins, chromosomal DNA,
excess primers, dNTPs, enzymes and buffer components/salts from the PCR reaction, residual organic
chemicals, and detergents. The quality of the DNA template for sequencing can be assessed by agarose
gel electrophoresis where purified DNA should run as a single band on the gel.
7.5 PCR Product Purification
Purification of PCR product is essential for optimal sequencing results. Unused primers and nucleotides
as well as salts should be eliminated prior to sequencing for optimal results. Dye-terminator sequencing is
particularly sensitive to the presence of excess amplification primers. Both forward and reverse primers
can act as sequencing primers, resulting in the generation of multiple labeled sequence ladders within a
single reaction. These additional fragments cannot be distinguished from the intended target, making it
impossible to interpret the sequence data. In addition, the excess primers and nucleotides carried over
from the PCR reaction can alter the ratios of reagents in the sequencing reaction sufficiently to
compromise both the fluorescent signal and the reaction fidelity.
A variety of approaches have been developed for the purification and sequencing of PCR products,
including dilution for direct sequencing, organic precipitation, column purification, gel purification, and
exonuclease I/shrimp alkaline phosphatase (ExoI/SAP) treatment. Each approach should be evaluated
based on the requirements of the specific application.
7.5.1 Dilution of PCR Product
This approach is useful in those cases where the PCR reaction has been rigorously optimized to use
minimal concentrations of primers and nucleotides without interfering with the efficiency and fidelity of
amplification. The PCR product can be diluted 1:5 or 1:10 in highly purified water (i.e., usually by a
combination of techniques that can include carbon adsorption, distillation, deionization, reverse osmosis,

Number 40 NCCLS
ultraviolet radiation, and other methods) and used directly as sequencing templates. Even though this
approach will require no manipulation of the PCR product before sequencing, it will require extensive
optimization to ensure that the sequencing reaction delivers sufficient quality sequence data. This requires
that the PCR reaction is optimized regarding primer and nucleotide concentration, but also to avoid
generation of nonspecific amplification products that can interfere with the quality of the sequencing data.
7.5.2 Gel Purification
Agarose gel electrophoresis separates PCR reaction products and byproducts by size. After gel
electrophoresis, the desired PCR product can be excised from the gel and extracted by a variety of
different methods, including commercial products such as columns.23,24 The primary advantage of gel
purification is that the specific PCR product can be separated from the byproducts that are generated
during the amplification process such as nucleotides, primers, and any other nonspecific amplified
product. The major disadvantage of this process is that ethidium bromide staining and UV illumination
are used to identify the desired reaction product on the gel and this process can nick the PCR product that
will subsequently be used as sequencing template. Furthermore, this process is time consuming, produces
low yield, is not amenable to automation, and most importantly, is not efficient in separating DNA
fragments based on size, so some residual byproduct may remain. In addition, there are some agarose
products that contain small molecules that can interfere with the sequencing enzymatic reactions.
7.5.3 Column Purification
There are a number of commercially available products for PCR product purification. The most widely
used product uses a silica gel membrane that binds DNA preferentially and allows elimination of unused
primers and salt-eluting DNA into very small volumes such as 50 µl.25,26 Most of these columns have
different cutoff values based on the size of the DNA molecules. For example, some columns will separate
oligonucleotides 40 bases or shorter from the PCR product, allowing nucleotides and primers to be
eliminated. Ultrafiltration columns are other commercially available options. Ultracentrifugation columns
allow separation based on molecular weight, for example, there are columns that have a 100-kDA cutoff
and consequently can be used to separate the desired PCR products from single-stranded oligonucleotides
of less than approximately 300 bases in length and double-stranded DNA less than 125 base pairs in
length. Other commercially available columns use gel filtration chromatography to preferentially bind
small DNA segments, allowing elution of larger product for sequencing. One of the major advantages of
column purification is that the procedure is extremely rapid, simple, and amenable to automation. In
addition, it has been shown that these products have a high degree of reproducibility. It is important to
point out that byproducts generated during amplification with similar size to the intended amplicon will
not be effectively removed using these procedures and these may interfere with subsequent sequencing.
Careful optimization of the PCR reaction to avoid these byproducts should alleviate this problem.
7.5.4 ExoI/SAP
Another method for PCR product purification for sequencing is using an enzymatic reaction. Following
PCR, treatment of PCR product with an ExoI degrades single-stranded oligonucleotide DNA such as
unincorporated primers.27,28 Furthermore, treatment with shrimp alkaline phosphatase (SAP)
dephosphorylates residual oligonucleotide primers and as a result, they become inactivated. Following
treatment, the residual enzymes can be heat inactivated and the enzymatic products can be directly used
for the sequencing reaction. The major advantage of this procedure is that it is simple, cost effective, and
can be easily automated for high throughput applications. Furthermore, this method is highly reliable. The
major disadvantages of this procedure are that this reaction also needs to be optimized for the best
possible performance and that secondary byproducts of PCR reaction will not be removed. Consequently,
the PCR must be optimized to prevent formation of byproducts.

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7.6 Quantity and Quality of DNA Template
Another factor that has a major impact on the quality of the sequence data is the quantity and quality of
DNA target used in the sequencing reaction. The quantity of DNA is typically determined by
spectrophotometry at 260 nm, fluorometry, or electrophoresis.
The quantity of DNA used for the sequencing reaction will depend upon the sequencing chemistry used
and the manufacturer of the reagents. It is advisable to review the manufacturer’s recommendations as to
the amount of DNA required to obtain good sequencing data. In general, the amount of template will
depend upon the PCR product size, double-stranded versus single-stranded template, etc. As a point of
reference, PCR products between 100 to 200 bp will require 1 to 3 ng of template, and products between
1000 to 2000 bp will require 10 to 40 ng. If too much template is used, one can obtain very strong initial
peak heights that fade rapidly as the sequencing reaction progresses. This can occur due to depletion of
the dye label early in the reaction. On the other hand, if too little template is used, peak height and
strength will be reduced, making data analysis difficult.
8 Sequencing
8.1 Sequencing Reactions
The technology employed in most sequencing is a combination of enzymatic chain termination with
dideoxynucleotides29 and cycle sequencing with thermostable DNA polymerases.30,31 In general,
sequencing reactions require the following components:
• sequencing primers;
• sequencing mix containing mixtures of deoxynucleotides and dideoxynucleotides and buffer;
• thermostable DNA polymerase;
• dye label (either the sequencing primers or the dideoxynucleotides can carry the label); and
• DNA template (see Sections 5 and 6 of this document).
8.1.1 Sequencing Primers
Refer to Section 7.1 for information on designing sequencing primers.
8.1.2 Sequencing Mixes
Sequencing mixes vary depending on whether dye-labeled primer or dye-labeled dideoxy-terminator

chemistry is used.
When using dye-labeled primers, it is necessary to have four separate sequencing mixes for A, C, T, and
G. There are multiple approaches to using dye-labeled primers. The simplest uses a single primer labeled
with one dye. Each mix would contain dATP, dGTP, dCTP, and dTTP and one of the dideoxynucleotides
ddATP, ddGTP, ddCTP, or ddTTP, formulated in ratios optimized to produce maximum read length and
accuracy. This is because all the fragments will have the same dye label and it would be impossible to
differentiate the sequencing DNA fragments terminated with each dideoxynucleotide if they were all
included. Each sequencing mix should be analyzed separately on a lane in a gel or capillary, and the data
for all four sequencing mixes should be combined by the sequencer software to give the complete
sequence.

Number 40 NCCLS
Another approach is labeling the same primer separately with four different fluorescent dyes. This results
in four dye-labeled primers, sharing the same DNA sequence but having different dye labels. Sequencing
ladders are generated in four separate base-specific reactions for A, C, T, or G. The products from these
reactions can be combined and prepared for electrophoresis.
The forward and reverse sequencing primers can be labeled with different dye labels therefore allowing
forward and reverse sequencing to be performed in the same reaction tubes. In this approach, a separate
reaction is required for A, C, T, or G. Each reaction is analyzed in a separate gel lane or capillary.
In dye-labeled dideoxy-terminator chemistry, ddATP, ddGTP, ddCTP, and ddTTP are all labeled with
fluorescent dyes. The sequencing mix would contain dATP, dGTP, dCTP, and dTTP and limiting
quantities of all the dye-labeled dideoxynucleotides ddATP, ddGTP, ddCTP, and ddTTP. The sequencing
primers should be unlabeled and the sequencing DNA fragment should be labeled when a dye-labeled
ddNTP is incorporated. This allows the sequencing reactions for A, G, C, and T to be performed in one
reaction tube and analyzed in one lane on a gel or a single capillary. The sequencer software analyzes the
data and generates a complete sequence from one lane. It is not possible to use multiple primers in a
sequencing reaction, so forward (sense) and reverse (antisense) sequencing must be done in separate
reaction tubes.
The advantages and disadvantages of dye primer and dye-labeled dideoxy-terminator chemistries for
sequencing are summarized in Table 1.
Table 1. Advantages and Disadvantages of Dye Primer and Dye-Labeled Dideoxy-Terminator

Chemistries for Sequencing
Chemistry Advantages Disadvantages
Dye Primer • More even signal intensities • Four reactions needed/less
• Less or no clean-up needed efficient
before electrophoresis • Compressions are possible
• Longer read lengths • More pipetting steps to set up
• Fewer structure-related stops reactions
• Labeled primers are available
for common vector binding
sites
Dye-labeled dideoxy-terminator • One reaction needed/efficient • More structure-related stops

• Compressions are resolved • Shorter read lengths
• Clean-up required before
electrophoresis
• Fewer pipetting steps to set
up reactions
• Unlabeled primers used
8.1.3 Thermostable DNA Polymerase
There are many possible thermostable DNA polymerases to use in cycle sequencing. Special
consideration is needed for selecting the enzyme for fluorescent dye-labeled dideoxy-terminator
chemistry. Enzymes with mutations that decrease the discrimination between dNTPs and fluorescently
labeled ddNTPs are preferred.32,33 Improved performance in dye-labeled primer sequencing can be
obtained by including a thermal stable pyrophosphatase to eliminate problems with pyrophosphorolysis.34

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The choice of enzyme will determine the appropriate ratio of dNTPs to ddNTPs that would be needed in
the sequencing mixes. Commercial mixes are available that are optimized for some of the DNA
polymerases.
The key performance selection criteria for an enzyme should be the combination of equivalent efficiency
of incorporation of the A, G, C, and T dideoxynucleotides leading to uniformity of band intensity for
sequencing DNA fragments, the number of bases sequenced, and sequencing accuracy.
8.1.4 Dye Label
The key selection criteria for a dye label are that it is compatible with the sequencing chemistry and
supported by the sequencing platform used. Commercial sequencing platforms have fluorescence
detection systems that are optimized for certain excitation and emission wavelengths. These specifications
will indicate which dyes can be detected and analyzed. Standards containing the dyes should be used to
calibrate the sequencing platform. This is especially critical when using a fluorescent, labeled dye
terminator chemistry, when four dyes are present in each lane or capillary.
8.2 Controls and Reference Standards

There are many steps between isolation of DNA or RNA and the final sequencing results. It is ideal to
have both a control that is taken through the entire process from DNA isolation, through PCR and
sequencing with a known sequence, and a reference standard that can be taken directly through the
sequencing reaction.
The results for the control and the reference standard can be compared to a set of specifications derived
during the verification of the sequencing assay. If the results of the control fail to meet specifications and
the results of the directly sequenced reference sample pass specifications, then the troubleshooting can be
directed to the steps prior to sequencing. If both fail specifications, initial troubleshooting should start at
the sequencing step.
Controls should resemble as much as possible the sample to be sequenced. It is important that the control
be stable and consistently prepared so that the results can be compared to the expected outcome. If the
assay is designed to test for polymorphisms or mixtures, then the control should have a polymorphism or
mixture as well.
Laboratories can obtain control materials from a variety of sources. Cell lines, clones, or reference
samples are available from commercial suppliers such as Coriell Cell Repositories
(http://locus.umdnj.edu/ccr/) or ATCC®a (www.atcc.org). Controls can also be well characterized clinical
specimens if properly consented from one’s own or from another laboratory. A sequencing standard is
usually available from the sequencing system manufacturer, since this is needed for the installation and
qualification of the system by the service technician. (Further information on how to use controls and
sequencing standards for quality assurance of sequencing assays can be found in Sections 8.6 and 9.1.)
8.3 Assay Verification

Each new sequencing-based assay must be verified prior to using the assay to generate reportable clinical
results. The assay should be subjected to analytical and clinical correlation studies to:
• characterize the locus/mutation(s) being detected (literature review);
• establish the performance properties of the assay to ensure the assay’s ability to provide consistent
and reliable results;
a
ATCC is a registered trademark of American Type Culture Collection.
Number 40 NCCLS
• establish the clinical utility of the assay (i.e., that the assay will measurably contribute to the
diagnosis of a disease or genetic status of the individual or family member tested);
• define aspects of the procedure which should be carefully regulated to maintain assay performance;
and
• define relevant limitations of the assay.
Such verification is necessary to ensure the safe and effective use of an assay for its intended use.
Detailed protocols for assay verification are beyond the scope of this document. Each laboratory should
develop its own verification protocols. Additional information covering assay verification is available in
NCCLS document MM1—Molecular Diagnostic Methods for Genetic Diseases and in the Standards and
Guidelines for Clinical Genetic Laboratories, 2003 Edition (American College of Medical Genetics
[ACMG]; www.acmg.net).
Sequencing-based assays address either detection of a sequence variation where the mutation to be
detected is known, or detection of a sequence variation from a reference or consensus sequence where the
sequence variation might be anywhere in the region sequenced. In both cases, mixtures of sequences
could be present (see Section 8.6.2 for additional information). The laboratory must identify and
characterize samples that are positive for the known sequences to be detected or representative of the
sequence variations to be expected. Depending on the rarity of the disorder, acquiring sufficient samples
for verification studies may be difficult. Samples may have to be obtained from other clinical laboratories
or researchers who study the disorder. Getting appropriate informed consent to use the samples in
verification studies is recommended. These samples should be verified by testing by alternate methods
such as allele or sequence-specific PCR amplification, or sequencing by other laboratories. The sequence
of interest can be cloned from a clinical sample and multiple clones can be sequenced to verify the sample
sequence. In addition, negative samples that have the expected reference or consensus sequence should be
obtained in order to fully validate the assay for sensitivity and specificity.
It is also important that analytical parameters that might affect assay performance such as instrument run-
to-run performance, different reagent lots, amplification and sequencing method variability, and operator-
to-operator differences are addressed.
8.4 Instrument Setup
Both gel and capillary sequencers are complex instrument systems. For clinical applications, the
instrument performance should be validated by installation and operation qualifications. The key aspects
are to ensure that the instrument is properly installed and performing as specified by the manufacturer.
Critical systems to address in the installation are:
• all the components are installed properly;
• power requirements are adequately met including consideration of noninterruptible service;
• ventilation requirements are met; some sequencers generate significant waste heat that must be
exhausted from the laboratory; and
• computer and software also require conditioned power and suitable back-up.
Operational qualification incorporates running a sequencing reference standard multiple times and
assessing that operators of the sequencing system are properly trained to maintain the equipment. All

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appropriate sequencing results must be obtained. Most sequencing systems are installed and tested by the
manufacturer as part of the purchase.
It is important that the sequencers receive required cleaning and preventive maintenance to function
properly. Many systems in the sequencer are not accessible to the user and require calibration and service
by the manufacturer. Proper alignment and function of the optical detection system is critical to high
quality sequencing results. A service contract is essential for maintaining a sequencing instrument, and
having access to a telephone at the instrument is valuable in troubleshooting with technical service. Most
manufacturers offer training courses for operating their equipment and software. The cost of training of
operators should be included as necessary in the purchase of the instrument. An operator should be
qualified by assessing performance on controls and reference standards prior to running test samples.
8.5 Electrophoresis
Electrophoresis on gel and capillary sequencers is controlled by the instrument’s software. There are
major differences between gel and capillary sequencers on the methods for preparing the separating
medium and loading the samples. Capillary sequencers automatically load the separation medium in the
capillary and load the sample. For gel-based sequencers, users have to cast a gel and load the samples
manually.
8.5.1 Gel-Based Sequencers
The quality of the gel is a major contributor to the overall quality of the sequencing results on gel-based
sequencing systems. Gels need to have uniform polymerization, no air bubbles, and the glass plates have
to be free of lint and light scattering materials. It takes training and experience to consistently cast high
quality gels. Visual examination of the gel can discern problems with air bubbles and/or uneven
polymerization. Since multiple lanes are used on a gel, there is potential for some of the lanes to generate
unusable data. This will result in the need to rerun the sequencing reactions that were affected on a new gel.
8.5.2 Capillary Array Sequencers
Capillary array sequencers use the capillaries over and over again until the maximum number of runs
specified by the manufacturer is achieved. Each capillary is separately calibrated for the dyes used in the
sequencing reactions, so that the software can perform the multicomponent analysis to identify each of the
dye-labeled fragments. There can be variation among capillaries initially and over time, so recalibration
should be done periodically. Changing of the capillary array requires optical recalibration and revalidation
of sequencing performance. It is possible that a capillary will have a problem in a sequencing run that
causes the loss of sequencing data. An air bubble in the separation media, sample loading problems, or
other problems in the performance of a capillary could lead to a loss of data. These sequencing reactions
would have to be reanalyzed. Unlike gels, which can be visibly inspected for obvious defects, the loading
of the capillary is not easily observed. If your instrument system allows observation of the acquisition of
the raw data, problematic capillaries can be identified. The choice of polymer is defined by the
manufacturer of the sequencer and users should use the polymer as recommended by the manufacturer.
8.6 Data Examination
8.6.1 Sequence Quality Assessment
After electrophoresis is finished, the sequencing analysis software translates the electrophoretic data into
sequencing data. The first assessment of the quality of the sequencing run is whether the software was
able to generate a sequence for each sample. If multiple primers are used to sequence larger targets, the
ability of the alignment software to generate the consensus sequence from each of the reactions is a good
quality measure. Sequences should be cross-referenced to the electropherogram data by way of an output

Number 40 NCCLS
file from an automated instrument. The important characteristics to review in the electropherogram
include signal intensity (peak height), signal to noise ratio, clear spacing or resolution of the peaks, low
background, and noise (minor peaks). Regularity of base spacing may be compromised by G- or C-rich
regions. Background peaks should not exceed 5% of the peak height for a homozygous peak. When the
electropherogram characteristics are acceptable, then the instrument software is usually able to generate
high quality sequencing data. This is easily evaluated by looking at the number of bases called or not
called by the software. In evaluating the sequence, the range of accurate base calling should be known or
established. If the number of uncalled bases exceeds either the instrument manufacturer’s specifications
or a laboratory’s specifications derived from verification studies, then the electropherograms should be
visibly examined to determine whether the sample should be rerun. For automated instruments or
purchased kits, the manufacturer should be consulted regarding the range of accurate base calling. The
manufacturer’s manual for the sequencer and the sequence analysis software usually give examples of
problematic sequencing data and what to do. Additional information can also be found on the Internet at
http://seqcore.brcf.med.umich.edu/doc/dnaseq/interp.html.
In performing a sequence analysis to confirm a mutation, examination of sequence from both strands is
recommended. Sequencing of both strands may not be needed for all applications, but it is very helpful
when looking for heterozygous single nucleotide substitutions. When this is not practical, either the
quality of the sequencing analysis should be reviewed (see above) or the mutation confirmed by a repeat
analysis. Comparing the sequence of the sample to a reference sequence is a useful quality assessment
tool. A high level of concordance indicates that a sample was sequenced properly and the sequence is
from the target region. Since in most assays, only a subset of the sequence is important clinically, the rest
of the sequence provides an internal standard for the sequence quality. In many applications, only a small
segment of the sequence is expected to have variations, and most of the sequence can be compared to a
reference or consensus sequence. The “all base accuracy” can be calculated and used to assess the quality
of the sequence obtained. All base accuracy is calculated by determining for the total number of bases
sequenced for a sample the percentage of the bases called that agree with the expected base call in the
reference sequence. A subset of the sequence that is expected to be highly conserved between the patient
sample and the reference sequence should be chosen for the analysis. Base-calling errors are often not
evenly distributed throughout the sequencing run. The largest number of miscalls generally exists at the
beginning of the sequence.35 Laboratories should adjust their sequencing reactions to account for this by
beginning sequencing far enough upstream from the sequence of interest to minimize base miscalling for
the DNA sequence of interest. Differences between the sample sequence and the reference sequence can
be due to at least three causes: 1) The sample has a different sequence, either a polymorphism or a
mutation, when compared to the reference or consensus sequence; 2) The infidelity of the enzymes used
for PCR generated a base difference between the sample and the reference or consensus sequence; or 3)
The automated sequence analysis software analyzed the electropherogram incorrectly, and has called an
incorrect base in the sample. The expected level for all base accuracy depends on the amplification and
sequencing chemistry used and the sequencing equipment chosen.
The role of the operator in getting high quality sequencing results should not be underestimated.
Operators need to be thoroughly trained in executing the assay steps, operating the instrument, and
interpreting the sequencing data. Operators should be qualified before performing the assay and
proficiency testing should be employed.
8.6.2 Detection of Sequence Mixtures
When sequencing-based methods are designed to detect a mixture of sequences that may be in the sample,
such as mutations in a background of wild type sequence, it is essential that the capability of the assay to
detect mixtures is determined. The first step is the acquisition of reference samples or materials that can
be used to generate samples that have known mixtures of sequences, for example, Sequence A or
Sequence B. These samples must be taken through the entire method, including sample preparation, and
analyzed with the methods to be used on patient specimens. Sufficient samples and replicates of defined

Volume 24 MM9-A
mixtures should be analyzed to determine the level of mixture (i.e., 30% of Sequence A with 70% of
Sequence B) that can be reproducibly detected within a 95% confidence interval. It is important that all
the variations in the method, such as instrument run-to-run performance, different reagent lots,
amplification and sequencing method variability, and operator-to-operator differences, are addressed in
the study.
Sequencing in both directions can add confidence to the base call of a mixture. However, if highest
sensitivity is desired, a mixture may be evident in one direction and not confirmed in the other. The cause
of this may be the differences that sequencing enzymes have in incorporating the different ddNTPs into a
DNA strand, thus the base peak in one direction will be strong enough to call, but not strong enough in
the opposite direction. Another possibility is that the noise in the sequence data is higher for one direction
than the opposite direction, and the mixture is above the noise in one direction, but not in the other. If the
highest possible sensitivity is important, then the verification study should incorporate single direction
mixture determination in the analysis. The confidence level should be determined for both single and bi-
directional sequencing sensitivity.
9 Data Analysis
9.1 Sequence Review
9.1.1 Quality Assurance and Quality Control
Sequence analysis for clinical purposes is performed to either confirm a previous finding or determine the
sequence of a defined region of DNA. Methods for estimating error differ for each purpose. This can be
appreciated in considering what an error rate of 1% means for each purpose cited. In confirming a
previous finding, an error rate of 1% tells us that 99 out of 100 times we will expect the correct answer.
An error rate of 1% in sequencing a 500 base pair fragment is expected to result in five incorrect base
determinations.
Sequence controls should include a wild-type specimen having characteristics of actual test samples.
Results from sequencing this control should be available for comparison to results from test samples and
also useful for training operators. The wild-type sample should be run at intervals sufficient to detect
changes in performance of the platform and consistent with current regulatory requirements.
Either reverse strand sequencing or a duplicate repeat of the sequencing reaction should be performed,
even when a reference sequence is used. Sole use of a reference (control) sequence is not always a good
quality assessment tool since it may be of higher quality than the clinical sample.
Quality assurance and control measures for sequence analysis can be broadly divided into two categories.
The first covers efforts to ensure appropriate sample collection, preparation, and chemistries, and the
operation of the technology platform according to specifications. The second includes efforts to ensure
that the correct sequence is determined from the raw data generated. Interpretation of raw data is often
performed through a variety of software tools. Operator review of this interpretation is critical to
determine whether the data returned are reasonable or if problems may have occurred in the sequence
analysis or software interpretation.
9.1.1.1 Software Interpretation
Raw data obtained from a sequencing analysis represent mobility shifts of nucleic acid fragments though
a matrix (i.e., acrylamide). For acrylamide gels, in which a number of samples may be run
simultaneously, the software must be capable of accurate lane tracking and assignment. Problems can
occur if lanes are smeared, streaked, pinched, tilted, or wavy. Direct operator review of the raw data (the
actual gel or computer recreation) is sometimes necessary to determine whether any of these conditions
Number 40 NCCLS
exist. Further, the mobility of fragments depends on their size and specific dyes used in the detection
process. Dyes can alter mobility shifts. Software uses mobility-shift files that contain data to compensate
for changes in mobility due to the presence of dyes. Sequence platforms are often flexible and can
accommodate multiple dye combinations. Use of the correct mobility-shift file is necessary for correct
software interpretation of the results.
Laboratories performing diagnostic sequence analysis should be able to document the quality of sequence
determinations. A number of software packages provide “quality scores” useful for this purpose. Many
factors are often included in the calculation of a “quality score” and may include consideration of:
• Signal strength – This refers to the peak height and may be expressed in relative units or normalized
against some standard (electronic or control sample) sequence.
• Signal-to-noise ratio – The ratio of an actual signal (peak) to other signals (peaks) in the same
vicinity.
• Overlap – The extent to which adjacent peaks occupy the same or different positions.
• Loss of signal intensity – The extent to which signal strength is lost during the course of a sequence
analysis.
• Baseline variation – The extent to which the baseline remains at a constant signal strength. The
signal strength may be absolute or relative to normalization against some standard (electronic or
control sample).
• Compression – Changes in the relative spacing between subsequent peaks.
Although quality scores are useful in documenting the overall quality of a sequence determination, the
composite score may or may not reflect certain problems. For this reason, operator evaluation of both the
raw and processed data is critical in assuring the quality of the procedure. The sequence should be
visually evaluated to determine if any of the items listed above adversely affect the interpretation of the
raw data.
Samples to be sequenced may contain mixtures of nucleic acids. These may or may not be expected but
their presence must be evaluated through the software and visual inspection of the data by the operator. A
mixture may result from:
• heterozygosity;
• mosaicism;
• fragments resulting from amplification of pseudoalleles;
• spiking with control materials;
• other multiple amplification products (intended or unintended); and
• contamination.
9.1.1.2 Examples of Problems in Interpretation of the Raw Data
It is important for the operator to understand and recognize the range of analytic outcomes that are
clinically reasonable or may be attributable to problems with the technology platform or software.
Problems and their causes can often be inferred through evaluation of the original or recreated mobility-
shift pattern (i.e., the acrylamide gel), the raw unprocessed data, the processed data, and the quality score
and indicators provided by the software used. It is also important to note agreement or no agreement
between each strand when reverse strand sequencing is performed on duplicative samples. The quality of

Volume 24 MM9-A
sequence determined for the wild-type control may also be useful as a comparison in identifying
problems.
Poor sequencing data can have several causes. The data may show:
• no recognizable signal;
• poor lane recognition and tracking;
• noise resulting in missed or incorrect base calls;
• signal loss after base-calling begins; and
• unexpected stops.
Potential causes for these problems may include:
• poor template (i.e., low concentration or poor preparation);

• inadequate purification of template prior to sequencing reaction;
• poor primer and template annealing;
• multiple annealing sites for primer;
• inappropriate concentration of template or primer;
• failed PCR amplification;
• template different from what is expected (i.e., due to unknown genomic sequence variation or
unanticipated RNA processing);
• contaminating sequences present in reaction mix;
• poor primer design or purity (low Tm, primer-dimer formation);
• interfering secondary structure within the template;
• reagents not performing as expected or wrong concentrations used;
• wrong mobility file used; and
• low signal strength resulting in failure of software to interpret raw data.
Software interpreted sequence data may on occasion appear to indicate a mixture when in fact, a
homogeneous sequence is present. This can occur if more than one primer is present in the sequencing
reaction, a secondary hybridization site, or secondary structure within the template.
Changes in the operating characteristics of the nucleic acid sequencer while data are being collected can
result in aberrant results. Use of a standard control during the sequence run provides one measure that the
technology platform is operating as expected.
Troubleshooting low-quality sequencing runs can be complex and may involve investigating multiple
possible causes. If unable to resolve the problem, the operator should consult the manufacturer or seller of
the sequence reaction kits or technology platforms to help identify and fix difficulties.
Software tools exist that are both platform dependent and independent. PHRED is one prototypic software
program able to estimate the likelihood of error based on peak mobility and shape analysis.36 This
software package can be adapted to a number of sequence chemistries and technology platforms. PHRED
scores are reported as 10x the log of likelihood of error. A PHRED score of 40 or higher is appropriate for
sequencing a 1000 base pair fragment. This score means there is a 1/10 000 likelihood of error. PHRED
was primarily designed as a research tool and is cumbersome to integrate into a clinical environment.
One complexity of PHRED is its requirement for look-up tables that take into account gel formulation and
sequencing chemistries. Commercial software packages have been developed more amenable to
implementation in clinical laboratories. These programs are available from manufacturers of sequencing
instrumentation that support their equipment as well as independent vendors that have developed
programs useful in analyzing data from a number of sequencing platforms. Laboratories performing

Number 40 NCCLS
diagnostic sequence analysis should use one of these programs to determine the sequence and as an aid in
documenting the quality of the sequence run.
9.1.2 Sequence Editing
Any sequence editing that differs from what is reported by the software interpretation needs to be
documented and justified. Included in the documentation should be data, a description of the data
examination process, and a clear explanation for the decision made to edit the sequence. It is advisable to
have the decision to edit the sequence reviewed by a second person familiar with the sequencing
methodology used.
9.2 Clinical Interpretation
Results should be interpreted in the context of the patient’s clinical findings, other laboratory tests, and
findings from the sequence analysis. Relevant information such as the prevalence and frequency of
sequence variations found, family history, inheritance pattern, factors related to gene expression, and
clinical significance (see Section 9.2.2) should be considered in deriving an interpretation. Sequence
findings, normal variations, and mutations should be referenced to listings in GeneBank, other DNA
sequence databases, or the published literature.
In developing an interpretation, confounding factors may impact on the accuracy of the sequence
determination. These may include allele dropout (due to amplification artifact or primer-binding site
mutation), unexpected deletions, insertion, other rearrangements, mosaicism, or other interfering DNA
sequences. It is the responsibility of the laboratory performing the test not only to be aware of those genes
and disease conditions in which these events are more likely to occur, but to recognize that they can occur
in other situations as well. As appropriate, these factors should be noted and explained on the test report.
Software packages are available that provide clinical treatment recommendations based upon the
sequence determined. For instance, several packages are available that provide recommendations for HIV
antiviral therapy. While recommendations to assess the validity of such software are beyond the scope of
this guideline, discordance in the interpretation of sequence results has been reported among a number of
these software packages.37 Therefore, users (laboratory personnel and clinicians) must take steps to
understand the usefulness, reliability, and limitations of clinical interpretations generated by such
software.
9.2.1 Nomenclature
As sequence analysis becomes increasingly important in making clinical decisions, it is critical to have a
clearly defined nomenclature to avoid misinterpretation of the laboratory results. Nomenclature is
necessary to describe the region of the genome or other nucleic acids being investigated (i.e., the gene(s)
or relevant noncoding regions, episomes, rRNA) and a common language for describing the sequence
variations therein.
In 1996, the Ad Hoc Committee on Mutation Nomenclature recommended a standard system for
describing sequence variations for human DNA.38 This effort led to the development of The Human Gene
Nomenclature Committee under the auspices of the Human Genome Variation Society.39-41 This
nomenclature continues to evolve and changes are made public through publication. We recommend that
this system be the standard by which sequence variations are described. Some of these recommendations
have been described in NCCLS and other publications and salient points are outlined below.42,43

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9.2.1.1 Reference Sequence
A single reference sequence should be established or referenced. A genomic reference sequence is

preferred to overcome difficult cases. However, if this is not available, a cDNA reference sequence
should be used instead. The laboratory report should indicate the nomenclature used in describing the
reference sequence. The standard for protein coding sequences is to have the nucleotide corresponding to
the A of the ATG start codon referred to as position +1. There is no nucleotide 0. The position preceding
the 5′ A should be designated as position -1. For mtDNA sequences, a standard reference sequence has
been published and is commonly used.41 Accordingly, the origin of heavy strand replication is designated
as the first base.
9.2.2 Clinical Significance
Clinical significance of a test should be based upon evidence that the sequence or variation thereof is
associated with disease, a predisposition to disease, or relevant clinical condition. In describing the
clinical significance of a human sequence variation, consideration must be given to prevalence rates in the
patient population and other epidemiological data.
In reporting the clinical significance of human sequence variations, it is important to describe its
relevance to the condition for which the test was referred. We recommend using the following categories
in describing the relevance of sequence variations. Sequence variations can occur in protein coding and
noncoding regions as well as regions that regulate RNA synthesis and other cellular activities. These are
adapted from guidelines adopted by the American College of Medical Genetics:44
• Sequence variation is previously reported and is a recognized cause of the disorder. Reported
when there exists credible documentation of a sequence variation and a clinical outcome.
• Sequence variation is previously unreported and is of the type which is expected to cause the
disorder. Insertions, deletions, and frame shifts can disrupt normal protein synthesis or regulation of
cellular processes such as transcription and translation.
• Sequence variation is previously unreported and is of unknown significance. These variations

represent base changes that do not change the coding sequence or effect known processing or
regulatory pathways. However, such sequence variations may still produce cryptic splice sites, affect
regulatory processes, interrupt exonic splicing enhancers and suppressor sites, or participate in other
mechanisms that may be associated with disease.
• Sequence variation is previously reported and is a recognized neutral variant. Reported when
evidence is available that the sequence variation has been consistently observed in a normal
population without association to disease or predisposition.
In cases where the outcomes of the finding of a sequence variation are not understood, family studies may
help clarify the clinical situation.
9.2.3 Limitations
Gaining useful information from sequence analysis is predicated on having an appropriate sample,
interrogating the appropriate region of the nucleic acid, and understanding the technical limitations.
Interpretation of sequence analysis may be limited by the following:
• Appropriate interpretation of sequencing results depends, in part, on knowledge of its relevance to the
medical condition referred. A clinically valid correlation between the presence of a particular

Number 40 NCCLS
sequence and a medical condition can be based on clinical, molecular, biochemical, and/or
epidemiological data. Low penetrance and/or variable expressivity may complicate the interpretation.
Other genes, sequence variations, and environmental factors may contribute to the medical condition
and should be considered in the interpretation.
• Some deletions, insertions, rearrangements, and other alterations may not be detectable by sequence
analysis. For instance, in humans, a deletion of the sequence under investigation in one chromosome
will not be obvious if only sequence analysis is used as the diagnostic tool. In such cases, a
heterozygous deletion may appear as a homozygous normal individual. Likewise, a rearrangement
may yield a correct sequence determination but not indicate that a recombination has taken place.
• Tissue-specific expression may impact the sequencing results and interpretation when RNA is the
starting material.
• Limited sensitivity may preclude detection of sequences present.
• Low titer, mosaicism, mitochondrial heteroplasmy, presence of other DNA sequences, or other
factors can limit the detectability of a particular sequence and should be considered in interpretation
of the results.
• Recent (whole) blood transfusions may confound the sequencing results and interpretation.
9.3 Reports
9.3.1 Critical Elements
Reporting of test results derived from molecular analysis has been described in NCCLS and other
documents and is applicable to DNA sequence analysis.42,45,46 Salient features include the name of the
individual, date of birth, specimen collection date, and other data. In addition to these, critical to the
reporting of results derived from sequence analysis, the following should also be included:
• The gene and/or chromosomal region interrogated should be clearly identified (CFTR gene, etc.).
• That part of the gene and/or chromosomal region interrogated should be specified (exons, splice sites,
etc.).
• The sequence and/or variation determined and position assignments should be specified and assigned
according to accepted nomenclature. Both the nucleotide and the codon where a sequence change
occurs should be indicated and frame shifts should be noted, including the codon position that results
in a stop.
• When appropriate, the corresponding change in amino acid sequence should be specified according to
accepted nomenclature.
• For human sequence determinations, results should indicate findings relevant to each allele.
• If available, other supportive data should be indicated that had been used in deriving the interpretation
(i.e., family history, other clinical findings, homology to related sequences).
• The clinical relevance of the findings (see Section 9.2.2).
• Analytic limitations (see above).

Volume 24 MM9-A
• Interpretive limitations of the analysis (i.e., a negative result does not rule out contributory mutations
present elsewhere in the genome).
• All databases used in the analysis should be referenced in the report. If applicable, a website reference
for identifying gene-specific databases should be included.
9.3.2 Confidentiality
All patient results should remain confidential and adhere to regulatory requirements. Generally, results
should only be made available to the referring healthcare professional. In some cases, results may be
communicated directly to the patient. In these situations, specific policies should be established to help
ensure that the patient understands the results, the limitations of the test, and recommendations for follow-up.
9.3.3 Preservation of Records
The laboratory should retain records for at least ten years and/or at least as long as mandated by
regulatory authority. In some cases, such as tests performed on newborns, it is advisable to maintain
records until the age of maturity.

Number 40 NCCLS
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Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications. Academic Press; 1990.
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Innis MA, Gelfand DH, Sninsky JJ. PCR Strategies. Academic Press; 1995.
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Niederhauser C, Hofelein C, Wegmuller B, et al. Reliability of PCR decontamination systems. PCR Methods Appl. 1994;4:117-123.
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Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339:237-238.
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Kitchin PA, Szotyori Z, Fromholc C, Almond N. Avoidance of false positives. Nature. 1990;344:201.
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Persing DH. Polymerase chain reactions: trenches to benches. J Clin Microbiol. 1991;29:1281-1285.
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Trower MK, Burt D, Purvis IJ, Dykes CW, Christodoulou C. Fluorescent dye-primer cycle sequencing using unpurified PCR products as
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Number 40 NCCLS
NCCLS consensus procedures include an appeals process that is described in detail in Section 8 of
the Administrative Procedures. For further information, contact the Executive Offices or visit our
website at www.nccls.org.
Summary of Consensus/Delegate Comments and Committee Responses

MM9-P: Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Proposed Guideline
Section 5.3, Specimen Quality Assurance
1. Quality Assurance seems incredibly brief and does not address as a guideline how to set up QA related to
sequence diagnostics.
• The subcommittee has reviewed Section 5.3 and has found that specimen quality assurance is
adequately covered. Quality assurance is addressed throughout the document and in significant detail
in Section 8.2, Controls and Reference Standards, and in Section 9.1.1, Quality Assurance and Quality
Control.
Section 8.2, Controls and Reference Standards
2. Positive and negative controls are not needed for every sequencing run, since the sequence itself is an internal
control.
• The subcommittee agrees with the commenter and it is not stated in the document that positive and
negative controls are needed for every sequencing run. However, it has been reported that the FDA has
required a company to specify running a control with every run for its FDA-cleared genotyping
product.
Section 8.6.1, Sequence Quality Assessment
3. Sequencing of both strands may not be needed for all applications, but can be helpful when looking for
heterozygous single nucleotide substitutions.
• The second paragraph of Section 8.6.1 has been revised with the addition of the commenter’s statement
as the second sentence.
Section 9.2, Clinical Interpretation
4. The issue of software generated clinical interpretations which interface with wet sequence software are not
addressed.
• Recommendations to assessing the validity of such software is beyond the scope of this guideline;
however, for informational purposes the following paragraph has been added in Section 9.2:
Software packages are available that provide clinical treatment recommendations based upon the
sequence determined. For instance, several packages are available that provide recommendations for
HIV antiviral therapy. While recommendations to assess the validity of such software are beyond the
scope of this guideline, discordance in the interpretation of sequence results has been reported among a
number of these software packages.37 Therefore, users (laboratory personnel and clinicians) must take
steps to understand the usefulness, reliability, and limitations of clinical interpretations generated by
such software.

Volume 24 MM9-A
NOTES

Number 40 NCCLS
The Quality System Approach

NCCLS subscribes to a quality system approach in the development of standards and guidelines, which facilitates
project management; defines a document structure via a template; and provides a process to identify needed
documents through a gap analysis. The approach is based on the model presented in the most current edition of
NCCLS document HS1—A Quality Management System Model for Health Care. The quality system approach
applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any healthcare
service’s path of workflow (i.e., operational aspects that define how a particular product or service is provided). The
QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The
quality system essentials (QSEs) are:
Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Service & Satisfaction
Personnel Process Control Assessment Facilities & Safety
MM9-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other NCCLS
documents listed in the grid, please refer to the Related NCCLS Publications section on the following page.
Purchasing &
Improvement
Organization
Management
Management
Information
Satisfaction
Assessment
Facilities &
Occurrence
Documents
Equipment
& Records
Service &
Personnel
Inventory
Control
Process
Process
Safety
X
MM6
MM5
Adapted from NCCLS document HS1—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, NCCLS document GP26—Application of a Quality Management
System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of three
sequential processes: preanalytic, analytic, and postanalytic. All clinical laboratories follow these processes to
deliver the laboratory’s services, namely quality laboratory information.
MM9-A addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section on the following page.
Preanalytic Analytic Postanalytic

Interpretation
Management
Test Request
Assessment
Laboratory
Collection
Specimen
Specimen
Specimen
Specimen
Transport
Post-test
Receipt
Review
Testing
Results
Patient
Report
x x x x x x x x
MM6 MM1 MM1 MM1 MM1 MM1 MM6 MM1
MM5 MM6 MM5 MM5 MM5 MM5
MM6 MM6 MM6 MM6 MM6
Adapted from NCCLS document HS1—A Quality Management System Model for Health Care.

Volume 24 MM9-A
Related NCCLS Publications*

MM1-A Molecular Diagnostic Methods for Genetic Diseases; Approved Guideline (2000). This document
provides guidance for the use of molecular biologic techniques for clinical detection of heritable mutations
associated with genetic disease.
MM5-A Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline (2003). This
document addresses guidelines for a variety of amplification-based laboratory tests, including polymerase
chain reaction (PCR), transcription-based amplification system (TAS), strand displacement amplification
(SDA), ligase chain reaction (LCR), and other methods now widely used in diagnostic hematopathology. This
guideline provides a basis for laboratory implementation and quality assurance in this important area of
diagnostic molecular medicine.
MM6-A Quantitative Molecular Methods for Infectious Diseases; Approved Guideline (2003). This document
provides guidance for the development and use of quantitative molecular methods, such as nucleic acid probes
and nucleic acid amplification techniques of the target sequences specific to particular microorganisms. It also
presents recommendations for quality assurance, proficiency testing, and interpretation of results.
*
Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should
refer to the most recent editions.
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CLMA FDA Center for Veterinary Medicine International Technidyne BC, Canada)
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National Association of Testing Bio-Rad Laboratories, Inc. – France Vicuron Pharmaceuticals Inc. Dr. Everett Chalmers Hospital (New
Authorities - Australia Bio-Rad Laboratories, Inc. – Plano, Vysis, Inc. Brunswick, Canada)
National Society for TX Wyeth Research Duke University Medical Center
Histotechnology, Inc. Blaine Healthcare Associates, Inc. XDX, Inc. (NC)
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Laboratory Standards (TCCLS) Copan Diagnostics Inc. (Belgium) Foothills Hospital (Calgary, AB,
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Laboratories Germany Anne Arundel Medical Center (MD) Geisinger Medical Center (PA)
BC Centre for Disease Control Dade Behring Inc. - Sacramento, CA Antwerp University Hospital Guthrie Clinic Laboratories (PA)
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MM9-A

Nucleic Acid Sequencing Methods in

This document addresses automated, PCR-based, dideoxy-terminator, and primer

Reproduced with permission, from NCCLS publication MM9-A—Nucleic Acid

Copyright ©2004. The National Committee for Clinical Laboratory Standards.

Area Committee on Molecular Methods

Subcommittee on Nucleic Acid Sequencing

Michael A. Zoccoli, Ph.D. Staff

Ira M. Lubin, Ph.D., FACMG

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

Summary of Consensus/Delegate Comments and Committee Responses............................................28

The Quality System Approach..............................................................................................................30

Related NCCLS Publications................................................................................................................31

Capillary electrophoresis, dideoxy-terminators, electrophoresis, gel electrophoresis, nucleic acid, primer,

Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine;

Antisense strand – Strand of DNA complementary to the sense strand.

2 An NCCLS global consensus guideline. ©NCCLS. All rights reserved.

Diagnostic/confirmatory testing – 1) Testing generally performed to evaluate the genetic status of

Electrophoresis – see Gel electrophoresis.

Extension/Elongation – The 5′ to 3′ synthesis of DNA starting from an annealed primer to generate a

Gel electrophoresis – Separation of molecules in an electric field within a matrix of agarose or

Oligonucleotide – A small polynucleotide generally synthesized on a machine; NOTE: Oligonucleotides

An NCCLS global consensus guideline. ©NCCLS. All rights reserved. 3

Sequence – The order of nucleotides (A, C, G, T, or U) in a strand of DNA or RNA.

Target sequence – Area of nucleic acid to be detected and sequenced.

4.2 Abbreviations and Acronyms

4 An NCCLS global consensus guideline. ©NCCLS. All rights reserved.

ddCTP dideoxycytidine 5′ triphosphate

5.1 Specimen Collection, Transport, and Storage

An NCCLS global consensus guideline. ©NCCLS. All rights reserved. 5

5.2 Specimen and Nucleic Acid Retention

5.3 Specimen Quality Assurance

6 Isolation of Nucleic Acid

6.1 Extraction of Nucleic Acid

6.1.1 Organic DNA Extraction Method

6.1.2 High Salt Method

6.1.3 Solid Phase Extraction Methods

6.1.3.1 Silica Technology

6.1.3.2 Glass Fiber Technology

6.1.3.3 Anion-Exchange Methods

6.1.4 RNA Extraction Procedures

An NCCLS global consensus guideline. ©NCCLS. All rights reserved. 7

6.1.5 Automated Nucleic Acid Extraction

7.1 Primer Design and Synthesis for PCR and Sequencing

• Primers should be at least 18 to 28 nucleotides in length to ensure good hybridization.

• Match Tm/Td of the primers to each other.

• Avoid runs of an identical nucleotide, especially four or more runs of Gs.

• Keep C-G content between 40 to 70%, preferably 50 to 55%.

• Design primers that are not complementary to each other.

8 An NCCLS global consensus guideline. ©NCCLS. All rights reserved.

• Avoid complementarity at the 3′ ends.

• Avoid complementarity at the 5′ ends.

• Avoid all known SNPs by checking primers against SNPs databases.

7.2 Amplification Parameters

An NCCLS global consensus guideline. ©NCCLS. All rights reserved. 9

• amount of starting template;