JCCLS MM6-A1（2017.10．1）

An Approved Guideline for the Quality Managementof Specimens for Molecular Methods (Part 2)
New Technologies and Sample Quality Control

Japanese Committee for Clinical Laboratory Standards (JCCLS).
2-7-13 Uchi-Kanda, Chiyoda-ku, Tokyo 101-0047 Japan

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CONTENTS（1）Introduction（2）Quality Assurance and Standardization Activities for Molecular Methods
1. Promoting Practical Applications of Genomic Medicine and Specimen Quality
Management Manual

2. International Standardization of Molecular Methods
2.1 International Standard ISO 15189 "Medical Laboratories - Requirements for Quality andCompetence"
2.2 International Technical Specifications Regarding Pre-examination Process
2.3 Various Standards for Pre-examination Process of Molecular Tests（3）Various Examination Technologies / Analytical Sample Quality Control

1. Chromosomal Analysis and FISH
1.1 Chromosomal Analysis of Tumor Cells (cancer diagnosis / tumor cells / living cells, mitotic
figures)
1.2 FISH (paraffin section) of Tumor Cells (cancer diagnosis / tumor cells / fixed cells)

2. Liquid based Cytology sample: Focusing on Cervical Cytology Examination
3. Array CGH
3.1 Blood
3.2 Tissues / Cells

4. Next Generation Sequencing (NGS)
4.1 Sample Quality Control for NGS Analysis
4.1.1 Blood
4.1.2 Tissues / Cells
4.2 DNA and RNA Quality Control for NGS Analysis
4.2.1 General Precautions on Preparation of DNA and RNA
4.2.2 DNA Quality Control
4.2.3 RNA Quality Control
4.3 Library Quality Control for NGS Analysis

24.3.1 General Precautions on Library Preparation
4.3.2 Quality Check of Fragmented DNA
4.3.3 Confirmation of Success or Failure of Adapter ligation and Quality Check
4.3.4 Quality Check of DNA library for NGS

5. Circulating Tumor Cell (CTC) Measurement (cancer diagnosis / peripheral blood / trace
amount cells)
6. miRNA · Exosome
6.1 microRNA (miRNA)
6.2 Exosome

7. Blood Circulating Cell Free Nucleic Acid
7.1 Blood Circulating Cell Free Nucleic Acid: Tumor
7.2 Noninvasive Prenatal Genetic Testing

8. Mitochondrial DNA（4）Appendix
3-1: Quality Control of DNA for Array CGH
3-2: Quality Check of CGH Microarray DNA Specimen * Agilent CGH / CGH + SNP Microarray
4-1: Quality Check of NGS DNA Library * SureSelect4-2: Quality Check of NGS DNA Library * HaloPlex
4-3: Quality Check of NGS DNA Library * Ion AmpliSeq
4-4: Quality Check of NGS RNA Library * TruSeq RNA Access（５）Bibliography
1. Literature
2. Guidelines etc.

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（1）Introduction
In molecular methods, to ensure accurate measurements, it is necessary to standardize
workflow processes in the pre-examination (pre-analysis) stage that have a potential togreatly influence the results. There are many points at which various properties of specimens
can affect nucleic acid extraction and the measurement process. However, methods forevaluating and avoiding the influences of sample preparation on results have not yet beenadequately established. Against this background, the WG-2 of Technical Committee onStandardization of the Gene based Tests in the NPO Japanese Committee for ClinicalLaboratory Standards proceeded with the development of a quality evaluation system forspecimens. Based on survey results regarding the actual situation in the field and obtained
evidence, a draft of the "Specimen Quality Control Manual for Molecular Methods" wasissued in the year 2009, and thereafter, an approved version of the document was released in
2011.
Analytical validity is greatly affected by the quality of the specimen. In securing analytical
validity, this manual plays an important role in the quality assurance of molecular methods
in the care of individual patients and in promoting the development and application of new
therapeutic reagents into the clinical practice. This manual lists the representative types ofthe specimens used in three categories of molecular methods (nucleic acid test for pathogens,somatic gene test, genetic tests of the germ-line). Preparation of these specimens,
requirements at the time of storage, transportation, processing, and collection of various
categories of specimens also have been clearly described.
This manual is a comprehensive and practical document that fills a void in the Japanesesetting regarding the quality of specimens for the purpose of quality assurance of molecularmethods. It is widely used at the national level at the Advanced Medicine Experts Conferenceof the MHLW, and seeks to be adopted as a set of standards for a facility acting in advanced
medical practice and receiving consignments from external medical institutions. Meanwhile,even in fields not described in the manual, progress in gene-based technologies have been
accelerated in the development from research to clinical application, and the use of molecularmethods for services is expanding (Table 1). Chromosomal analyses are not included in theframework of the molecular methods, but chromosomal analyses are handled in the same
way as genetic testing in the diagnosis of congenital disease. In addition, testing for prenatal
diagnosis based on molecular methods has become the subject of laboratory services in recent
years, and the number of testing requests is increasing. For hematopoietic tumors,chromosomal and genetic analyses are the basis of the disease type diagnosis according to
the WHO classification. Depending on the results of the chromosomal analysis, FISH or DNA

4analysis can be later requested using specimens that were stored in Carnoy's fixative (Acetic
acid: methanol = 1: 3). Appropriate management of samples is also important for quality
assurance of examination even in those fields not included in this manual.
As new areas for technical implementation and application, there are circulating tumor cells,
circulating cell-free nucleic acids in blood, fetal DNA in blood, microRNA, microarray
methods, gene expression profiling analysis, array CGH, whole genome sequencing and more.
Even when these methodologies are conducted for the purpose of research, the results could
be returned to patients for medical care. In the fields of gene-related testing technology,
development and clinical applications for laboratory use are being actively promoted. As aresult, in creating data on clinical validity and clinical utility based on analytical validity,
methods for quality control of specimens to ensure measurement accuracy are important.
In view of this background, the WG-2 Committee selected items to be added to the"Specimens Quality Control Manual for Molecular Analytic Methods." With regard to theselected items, based on the recognition that securing the quality of the specimen isimportant for securing the quality of the measurement results, the "recommended operation
method" for the preparation of specimens is presented, followed by (1) Inappropriate
conditions unsuitable for testing, (2) Cause, (3) Troubleshooting, and (4) Measures to avoid
inappropriate situations. This manual also has clarified the requirements at the time ofcollection of various specimens subject to molecular methods. The WG-2 has decided toproceed with the document development and to publish it as “Specimen Quality Control
Manual of Molecular Methods (Part 2).”

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Table 1 Items Requiring Additional Description in "Sample Quality Control Manual for
Molecular Methods"
PurposeSpecimen types

Target Sample of

Examination method

Interest
1. Cancer diagnosis

Peripheral blood

Trace cells

Blood circulating tumor cells

DNA(CTC) measurement

RNA

Blood

circulatingcell-freenucleic acid
Targeted sequencing

2. Prenatal diagnosis

Tumor tissue

Viable cells

Chromosomal analysis

Tumor cell

Mitosis image

Targeted sequencing

Fixed tissue / cell

FISH

(Cell block)Targeted sequencing

Trace DNADNA analysis of fetal origin

Peripheral bloodin maternal blood
Amniotic fluid

Viable cells

Villus

Mitosis image

Umbilical

cord

blood
3. Diagnosis ofWhite blood cell

mitochondrial disease

Muscle tissue

4. Whole genome
analysis

Fixed cell

Chromosomal analysis

FISH

Viable cells
Mitochondria DNA

Mitochondrial DNA analysis

White blood cell

Viable cells

Chromosomal analysis

Tumor tissue

Mitosis image
Viable cells / Fixed cell

Array CGH

/ DNA
DNA

Whole genome sequencing
MicroarrayRNA

RNA sequencing
Microarray6

（2）Quality Assurance and Standardization Activities for Molecular Methods
1. Promoting Practical Applications of Genomic Medicine and Specimen Quality
Management Manual
Molecular Methods have been developed for clinical applications based on advancements inanalytical techniques as well as elucidation of molecular pathological conditions of diseases
such as infectious diseases and cancer. In recent years, analysis of human genes has
progressed, and not only single gene diseases but also molecular pathologies with a genetic
component such as common diseases and drug response, in which genetic factors andenvironmental factors are intertwined, are being elucidated. The application of diagnosis,
treatment, and prevention of diseases by development and clinical application of Molecular
Methods is in progress. Personalized medicine is being promoted in the development ofmolecular targeted drugs and companion diagnostic drugs based on elucidation of individual
differences in drug reactivity and molecular pathological conditions of diseases. In addition,with the enforcement of the Act on Medical Care for Patients with Intractable/Rare Diseases,
diseases that are difficult to diagnose, and specific chronic pediatric diseases have greatly
expanded in number. As a result, in FY2008 medical fee reimbursement, D006-4 genetic test
insurance listings increased from 36 diseases to 72 diseases.
Next-generation analysis systems based on simultaneous multi-item analysis (multiplex
analysis) and comprehensive analysis, including Next Generation Sequencing (NGS), havebeen developed and are being used for clinical trials and patient diagnosis as a development
and deployment of new technologies. As the number of Laboratory Developed Tests (LDT)
using new analysis systems increase, there are various problems such as practical application,system implementation, appropriate use, and arrangements for social infrastructure. In theclinical use of these new analytical techniques, it is necessary to ensure the quality ofmolecular methods in response to patient needs and advancement of technology.
In recent years, domestic and overseas activities have been focused on standardization ofmolecular methods as a way of ensuring accuracy. Based on the interim report (July 2015) of
the Genome Medical Realization Promotion Council (established under the Health andMedical Strategy Promotion Headquarters, Health and Medical Strategy Promotion Council)
promoting the realization of genome medical practice, in November 2015, a task force topromote the practical application of medical care using genome information (Genome MedicalTF) was set up.

Discussions at the national level were held regarding the establishment ofsystems such as quality assurance and accreditation of laboratory performing molecularmethods, as the basis of genomic medicine. The final report was released in October 2016. A
policy proposal stated, "To ensure the quality and accuracy of molecular methods, it is7

considered that the required level of Japanese version best practices / guidelines specialized
in molecular methods is necessary, and based on the discussion at the task force, we should
consider and formulate concrete measures etc. from now on."The quality of molecular methods largely depends on the quality of the pre-examination
processes, and in particular, the analyte to be measured. The use of this manual is expected
to contribute to the improvement of the quality of molecular methods in Japan and theprovision of high-quality medical care / health care based upon it.

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2. International Standardization of Molecular Methods
In contrast to the first-generation molecular methods aimed at the detection andquantification of a single target of interest, in recent years, simultaneous multi-item analysis/ multivariate analysis or multiplex analysis and more comprehensive genome wide analysis
have been developed (Figure 1). Detection methods including multiplex PCR method, DNAmicroarray methods, differentiation of gene mutations using melting curve analysis ofamplified products after nucleic acid amplification, and the like are being used.
As a genome-wide analysis technology, NGS technology has begun to be used not only for
research but also for clinical sequencing with limited detection targets. With the elucidationand clinical significance of biomarkers in cancer and the development of molecular-targeted
therapeutics, the need for simultaneous multi-item detection or multiplex analysis isincreasing. Based on the biological elucidation of complicated disease pathology anddiagnostic algorithms, simultaneous biomarker measurements of multiple items may berequired for individual assessments. Gene mutations as specific biomarkers are mutually
exclusive. As an example, there are fusion genes of ALK, ROS1, RET in non-small cell lungcancer, and fusion (chimeric) genes associated with typical chromosomal reciprocaltranslocation in hematopoietic tumors. Also, there are various mutations (e.g. ABL1) in one
molecule or various molecular mechanisms of resistance (e.g. BRAF) that are used as indicesfor predicting the resistance to treatment of a molecular targeted therapeutics. A multimolecular-targeted drug (e.g. multi-kinase inhibitor, MKI) has also been developed andclinically used for the purpose of improving therapeutic effect and overcoming resistance. In
actual clinical examination, the use of multiplex analysis is expected, given the small amountof specimen available, and to achieve reductions in time and cost. As a result, paradigm shifts
have occurred in FDA approval, and the platform of multi-gene diagnostics and many drugs
is now being adopted over conventional one-drug, one-gene diagnostics (EGFR, KRAS,

BRAF ).
In multiplex molecular testing, quality control of nucleic acids to be used is important insecuring high performance measurement. Based on that recognition, the JCCLS TechnicalCommittee on Standardization of Gene-Related Testing, in cooperation with the Japan bio
Measurement & Analysis Consortium (JMAC), made a proposal on "International
standardization related to sample quality for multi-item gene analysis technology - In vitro
diagnostic medical devices - General requirements and terminology of quality evaluation of
nucleic acid for multiplex molecular testing -"at the Singapore plenary meeting (November2013) of the ISO / TC 212 Technical Committee (Clinical laboratory testing and in vitro
diagnostic test systems). The proposal was approved as a preliminary work item (PWI). This9

proposed standard was approved as a proposal for a new work item at the plenary meeting(Toronto) in November 2014 (ISO NP 21474) and approved by voting. At the plenary meetingof November 2016 Kobe), it has been renamed to ISO 21474 "In vitro diagnostic medical
devices - Multiplex molecular testing for nucleic acids - Part 1 - Terminology and general
requirements for nucleic acid quality evaluation" and is under discussion.

Clinical use

Research use

Multiplex(1) Single(3) Genome scale(2)Multiplex / multiple
items

Whole genome
DNA

Exome

Multiplex/multivariate

Sequencing

RNA

Transcriptome

Multiplex/multivariate

Genomic expression

DNA/RNA target

Profiling
Multiplex PCR / probehybridization

DNA microarray

Target MPS

Epigenetic

miRNA

Fig. 1 Development of utilization of genome information10

2.1 International Standard ISO 15189 "Medical Laboratories - Requirements for Quality
and Competence"
For standardization of Molecular Methods, it is necessary to standardize the technology to
be used, and to perform testing in clinical laboratories standardized in terms of quality,competency and the way they provide the results of the test. ISO / TC 212 (Clinical laboratory
testing and in vitro diagnostic test systems) of the International Organization forStandardization (ISO) 212, which has contributed to the quality improvement of clinicallaboratory testing and in-vitro diagnostic test systems, has emphasized the importance of
pre-analytic process in ensuring measurement accuracy. In this technical committee,
discussions and standard document development are now active, and international standards
for definition and procedures are being developed to ensure sample quality.
The international standard ISO 15189 ：2012 “Medical laboratories - Requirements for
quality and competence” was formulated as a standard for the evaluation of reliability andobjectivity in the clinical laboratory in ISO / TC 212. In its introduction, the significance ofthe primary sample (specimen) preparation as an object of standardization in a series ofprocesses of clinical laboratory services is clearly stated as follows. “Medical laboratory
services include arrangements for examination requests, patient preparation, patient
identification, collection of samples, transportation, storage, processing and examination ofclinical samples, together with subsequent interpretation, reporting and advice, in addition
to the considerations of safety and ethics in medical laboratory work.”. In the technicalrequirement of the standard document, regarding preparation of specimens in the preexamination process, items of 5.4.4 "Primary sample (specimen) collection and preparation ",5.4.5 “Sample (specimen) transport", 5.4.6 "sample (specimen) reception" and 5.4.7 "Preexamination- handling, preparation and storage" are developed in 5.4 "Pre-examination
process". While the international standard ISO 15189: 2012 provides chapter 5.4 "preexamination processes", the procedure description on patient safety and specimen quality
currently is inadequate.
2.2 International Technical Specifications Regarding Pre-examination Process
ISO / TS 20658 "Medical laboratories examinations - Requirements for collection, transport,
receipt and handling of samples" as a technical specification complementing therequirements of the pre-examination process of ISO 15189 has been proposed and relevant
documentation has been developed.
The chapter is divided into "Integration, Stability of the primary sample (specimen)","Transport of the primary sample (specimen)", "Receipt and evaluation of the primary sample

11(specimen)", and " Storage of primary sample (specimen) until testing" for the purpose of
securing the quality of specimens. It is stated that proper temperature, storage conditions
and time are important for ensuring the integrity of the primary specimen in the preservationof the sample and it is necessary to confirm the validity of these steps and periodical audit.
2.3 Various Standards for Pre-examination Process of Molecular Tests
Recently, remarkable progress has been made in molecular diagnosis and clinicalapplications using nucleic acids, proteins and metabolites in human tissues and body fluids.
The profile and integrity of these molecules will change in the pre-examination process,loweringthereliabilityofsubsequent

measurements.

In

Europe,the

SPIDIA(Standardisation and improvement of generic Pre-analytical tools and procedures for In-vitro
DIAgnostics) project formed by a consortium composed of 13 countries was established in2009 to standardize the work procedures of pre-examination process. Based on the outcomes,
standard documents were described and submitted to the CEN/TC 140 committee to develop
the European standards. In ISO/TC212, the series of CEN documents are currently underdevelopment as an international standard aiming at standardization and systematization ofpre-examination processes of molecular diagnostic testing (Fig. 2). The object of the standard
documents is three specimen types of formalin fixed paraffin embedded tissue, frozen tissue
and blood. That is, ISO 20166 (Molecular in vitro diagnostic examinations - Specifications forpre-examination processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 1:
Isolated RNA, Part 2: Isolated proteins, Part 3: Isolated DNA) respectively for RNA, protein
and DNA in formalin-fixed paraffin-embedded tissues, ISO 20184 (Molecular in vitro
diagnostic examinations - Specifications for pre-examination processes for frozen tissue - Part
1: Isolated RNA, Part 2 : Isolated proteins) respectively for RNA / protein in frozen tissues,and ISO 20186 (Molecular in vitro diagnostic examinations - Specifications for pre examination processes for venous whole blood - Part 1: Isolated cellular RNA, Part 2: Isolated
genomic DNA, Part 3: Isolated circulating cell free DNA from plasma) respectively for
peripheral blood cell RNA, peripheral blood genome and blood circulating free DNA in blood.
In addition to those developing tests in clinical laboratories and molecular pathology
laboratories, the intended users of these standards are supposed to be in-vitro diagnostic
device manufacturers, development and research institutions, and biobanks. (Figure 2).

12Collection

StorageTransportation

Processing

Nucleic acidextraction

Specimen collection (ISO/TS 20658)

Blood nucleic acid/ RNA/ genome (ISO 20186), FFPE (ISO20166) , Frozen tissue (ISO20184)

Molecular Methods

Specimen quality management manual (JCCLS)

Nucleic acidquality

Amplification

Detection

Multipurpose / multivariate(proposed in Japan)

ISO21474 “In vitro diagnostic medical devices – Multiplex molecular testing for nucleic
acids – Part 1 – Terminology and general requirements for nucleic acid quality evaluation

Fig. 2 Standardization activity of pre-examination process

13（3）Various Examination Technologies / Analytical Sample Quality Control
1. Chromosomal Analysis and FISH
1.1 Chromosomal Analysis of Tumor Cells (cancer diagnosis / tumor cells / living cells,
mitotic figures)
When venous blood is used as a sample, collect it with a heparin-containing blood collection
tube, thoroughly invert and mix the tube, and store it in a refrigerator. In the case of bonemarrow fluid, collect it in a dedicated preservation solution (RPMI 1640 culture medium or
the like) and store it in a refrigerator. Both cultures should be started within one day ofsample collection. Depending on the condition of the patient, adequate numbers of whiteblood cells may not be obtained, so specimens should be collected at an appropriate time.
(1) Inappropriate conditions for testing
1） Relevant cells undergoing mitosis may not be observed and it may be impossible tojudge an appropriate karyotype due to the disease or treatment (nucleated cells canbe extremely low in number after administration of anti-cancer agents), or due to themethod of preparing or preserving the sample.
2） There is a possibility that specimens will have a large proportion of cell components
other than the tumor cells as the target of interest, such as normal cells and nontumor cells, among the total cells in the specimen. This will result in difficulty ininterpreting the karyotype of target cells.
3） If a specimen is coagulated, it can interfere with establishing a proper culture.
4） Depending on the tumor cells of interest, their interphase mitosis images are notobtained at the time of chromosomal analysis and only normal karyotypes of mixed
normal cells can be observed.
(2) Cause
1） Sample collection was performed after administration of anti-cancer agent. Sample
preservation method was inappropriate, such as use of a frozen specimen.
2） Samples with low content of tumor cells were collected.
3） Anti-coagulant other than heparin was used. Alternatively, the admixture of
anticoagulant (heparin) was insufficient.
4） The division cycle of the target cancer cell is slow.
(3) Troubleshooting1） In some cases, cell proliferation can occur by culturing, but there is nocountermeasure when cells are poorly proliferating because nucleated cells die or are
scarce.

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2） There are no measures to avoid inappropriate situations.
3） Culture the cells after eliminating coagulation.
4） There are no measures to avoid inappropriate situations.
(4) Measures to avoid inappropriate situations
1） Collect specimens before anticancer agent administration. Comply with appropriate
storage conditions of specimens.
2） Collect sample areas with a high proportion of the tumor cells.
3） Collect with a heparin containing blood collection tube, and thoroughly invert andmix the tube after taking blood.
4） Perform FISH using interphase nuclei.

1.2 FISH (paraffin section) of Tumor Cells (cancer diagnosis / tumor cells / fixed cells)
In the diagnosis of some solid tumors, the FISH method using formalin-fixed paraffinembedded section (FFPE section) has been performed, meanwhile it is important to preparea specimen appropriate for the test, minimizing possible risk of influence by examination
procedure or operation.
As a recommended condition for fixation, 10% neutral buffered formalin is used and thefixing time is set to 6 to 48 hours. However, the penetration of formalin to tissue varies
depending on the type (such as how much fat is present), size, and shape, of the tissue to be
used, and thus the observation of the fluorescence signal is affected. Therefore, depending on
the specimen, it would be necessary to adjust the fixation time.
A thickness of 4-6μm is recommended for slicing of the tissue (the optimum thickness varies
depending on the cell size).
If the cancer cell to be observed has a large nucleus, and the sliced section is thin, thenucleus will be also sliced and thus will be only partially visible. If the section is thick, in thecase of a small nucleus, the nucleus becomes multilayered, making it difficult to observe andinterpret the patterns. Accordingly, it would be necessary to adjust thickness in considerationof the characteristics of the cancer cell to be observed and the probe to be used.
(1) Inappropriate conditions unsuitable for testing1)

No fluorescent signal is observed, and the judgment can not be made appropriately.

2)

The number of cells for counting is less than that required.

3)

A tissue is disrupted and thus can not be judged properly.

4)

A tissue is necrotic, and thus the fluorescence signal is not observed.

5)

A slice of tissue has peeled off from the surface of slide glass during heat treatment.

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6)

The background is too high.

7)

There are many fragments of nuclei, or there are many overlapping nuclei, and thus
proper observation is difficult.

(2) Cause
1)

Possible causes include autolysis of tissue due to long time from collection to
decalcification treatment and formalin fixation, improper fixation (excessive or
insufficient fixation) during formalin fixation, fragmentation of DNA due to not using
neutral buffered formalin (acid decalcification treatment), and degradation due toleaving sample for a long time after slicing.

2)

Biopsy material with few tumor cells was collected.

3)

Material was disrupted at the time of tissue collection.

4)

A necrotic portion is selected for the test material.

5)

An uncoated slide glass was used.

6)

A tissue is colored with a fluorescent dye, or a slide used has been insufficiently
washed or dried.

7)

The tissue section is not sliced at the appropriate thickness.

(3) Troubleshooting
1)

Overfixation may be improved by strengthening the digestion treatment of tissue tothe extent that the cell morphology does not collapse. In addition, when there aremany overlaps in the nucleus to be observed, reducing the thickness of the thinsection can strengthen the digestion of the tissue and improve it. There is notroubleshooting for tissue autolysis, insufficient fixation, fragmentation of DNA anddegradation of sample.

2)

There is no troubleshooting for biopsy materials with very few tumor cells.

3)

If there is a part that is not disrupted, use that part for analysis.

4)

If there is a part that is not necrotic, use that part for analysis.

5)

Coated slides should be used instead of uncoated ones.

6)

Observe the degree of fluorescence after the deparaffinization treatment, and if there
are background issues, adopt a decolorization procedure.

7)

Slice the tissue at a suitable thickness and re-stain.

(4) Measures to avoid inappropriate situations
1)

In preparing formalin-fixed paraffin-embedded (FFPE) samples, comply with the
precautions described in the HER2 examination guide (Breast Cancer HER2
Examination Pathology Working Group or Gastric Cancer HER2 Guidelines
Committee in the Japanese Society of Pathology), Guidance on ALK Fusion Gene

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Testing in Lung Cancer Patients（developed by the Japan Society of Lung Cancer),etc., and consider the preservability of the nucleic acid.
2)

Use a tissue section with a sufficient number of tumor cells as a sample.

3)

Avoid squashing tissues during collection.

4)

Do not subject a necrotic part of a sample to a test.

5)

Prepare a sample slide using a coated slide glass. If peeling is still observed, use anappropriate heating procedure (60-65 °C., about 30 minutes).

6)

Use non-fluorescent dyes and reagents that do not affect observation of fluorescenceof FISH. Clean the slide firmly in each step and pay attention to drying.

7)

Slice tissue at a proper thickness. Highly trained and skilled pathologists should do
the slicing.

2. Liquid based Cytology sample: Focusing on Cervical Cytology Examination
Liquid-based cytology (LBC) samples can be classified into gynecological ones and others by
the type of LBC vials. The uterine cervical cells are obtained with a brush and collected in apreservative solution prior to a smear sample being prepared.
Before and after the cytological examination, a nucleic acid amplification test (NAAT) for
detection of pathogens such as human papilloma virus, Chlamydia trachomatis, Neisseria

gonorrhea and Trichomonas vaginalis can be performed. NAAT should be performed on preprocessed aliquots of the specimen.
In addition, LBC are also utilized for fine needle aspiration biopsy specimens such as thyroid,
mammary gland, pancreas, body fluids specimens such as urine, pancreatic juice and bile
specimens, and pancreatic and biliary duct brush wash specimens, and others.
Depending on the manufacturer / provider, the collection method after obtaining the cervix cells
using a brush differs, for instance with or without the brush head in the solution of a vial. Although
it is less invasive, collection using cotton swabs in pregnant women is acceptable only if
doctors understand the risk of having far fewer cells in the specimen.
Immunohistochemical tests may be performed, especially in cases where it is difficult tomake a diagnosis by cytological examination.
When performing NAAT after the cytological test, handle the sample carefully to avoid
cross-contamination from others.
In addition, when an aggregate of cells is found, vortex or mix, as necessary, to disperse it.
As the preservative solution of the LBC sample is part of a medical device for preparing acytology sample slide, the appropriate storage temperature and period for the cytologicalexamination is described. Furthermore, NAAT should be performed within evaluated test

17

temperature and period for the stability of the nucleic acid.
Descriptions here are focused on the cervical cytology examination.
(1) Inappropriate conditions unsuitable for testing1） The target sequence (pathogen) is not retained.
2） The proportion of cancer cells is too low.
3） Excessive peripheral blood is obtained.
4） Excessive normal cells (mucus) are obtained.
5） Presence of substances that interfere with NAAT (contraceptive jellies and anti-fungal
medication).
(2) Cause
1） A sample is not stored a t the appropriate temperature and period of thepreservative solution for NAAT (nucleic acid of pathogen).
2） The presence and proportion of cancer cells can’t be confirmed immediately.
3） Brushing or fine needle aspiration cytology may obtain excess peripheral blood. In thecervical cytology test, blood may be present in the sample due to bleeding from alesion, menstruation and strong scraping.
4） Sampling without removing mucus, cervical cells are not scraped adequately, and thenumber of cells is reduced.
5） Obtain contraceptive jellies and anti-fungal medication.
(3) Troubleshooting1） Although the amount of nucleic acid at each stage of extraction, storage and transport
can be confirmed by an internal control derived from the human genome, this control
does not guarantee preservation of a nucleic acid sequence for detection of a target ofinterest (pathogen).
2） The presence / proportion of cancer cells is confirmed using methods such as acytological examination on the same specimen, specific gene expression (of epithelial
cells), FISH and the like.
3） Perform a hemolysis treatment and prepare the sample again. Check the extent ofblood volume and its effects. In the case of excessive blood affecting the test results,
re-collection should be also considered.
4） Use a nucleic acid extraction reagent and amplification / detection system which is
not affected by mucus contamination. Depending on the reagent to be used, resampling may need to be considered. Regarding the extracted nucleic acid, the proper
ratio of extracted amount to the cell number is confirmed by an appropriate internal

18

standard.
5） Estimate an inhibitory effect on NAAT by an internal standard, using contraceptive
jellies and antifungal creams.
(4) Measures to avoid inappropriate situations
1） Perform the test within validated conditions ( time and temperature) of thepreservative solution for NAAT (nucleic acid of pathogen).
2） Train a personnel for proper sampling methods to collect a sufficient number of cells.
3） Specimen collection at menstruation should be avoided. Collect specimens fromsuspected lesion.
4） Upon removal of mucus, cervical cells are taken with a brush. Train a personnel for
proper sampling methods.
5） Make sample collectors aware of the interfering effects of contraceptive jellies andanti-fungal medication in NAAT using LBC specimens.

3. Array CGH
Based on the quality control of pathological samples used for measurement by array CGH(comparative genomic hybridization), specimen collection, preservation, transportation andhandling should all be performed appropriately to obtain genomic DNA of recommended
quality.
The containers for transport of genomic DNA should be made of a material such as
Styrofoam, capable of maintaining a stable temperature inside and of preventing the collapseof tubes. The sample can be transported in refrigerated devices.
Samples may be stored in a container filled with dry ice for keeping at temperatures -70°C
or lower, when the examination is not scheduled for a certain time. Avoid repeated cycles offreeze and thaw. Compact tubes of DNase-free and low temperature resistant are used for
DNA samples.
3.1 Blood
Blood is collected with a blood collection tube containing an anticoagulant (e.g. EDTA), then
immediately mixed thoroughly by inverting. The tubes are stored refrigerated, if nucleic acidextraction is not performed on the day of blood collection.
Blood collected in EDTA-tubes can be stored at refrigerated for 3 days. Storage temperatureand transportation methods are selected based on the length of the period until DNA
extraction.
(1) Inappropriate conditions unsuitable for testing

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The quality of the extracted DNA is outside the criteria of the purity index. Severe
degradation in the extracted DNA will affect the results. Intact (high molecular weight)
DNA is desirable, whenever possible.
(2) Cause
The quality of the extracted DNA is influenced by the storage conditions of the sample,
DNA extraction method, DNA storage condition and handling.
■Assays using degraded DNA can result in a large error in results.
■Contamination with protein, sugar, lipid components, EDTA, and other impurities in the
DNA solution may affect the final result, for example, by reducing the efficiency offluorescence labeling.
(3) Troubleshooting
When the quality of the recovered DNA is evaluated and judged as unsuitable for CGH
analysis, perform re-examination from the DNA extraction step. If the sample itself is ofpoor quality, do the re-examination from the step of re-sampling.
(4) Measures to avoid inappropriate situations
In order to obtain genomic DNA suitable for array CGH, store the specimen/DNA sample,
process and extract the DNA using appropriate conditions and methods.
3.2 Tissues / Cells
The samples confirmed to substantially contain tumor cells are used, after performing
cytology and pathological examination on a split part of the samples, for the purpose ofascertaining the abundance ratio of tumor cells. Normal cells (non-tumor cells) are removed
from the tissues by microdissection as needed. The samples are handled appropriately,
depending on the needs of the target of sample, using suitable conditions of collection, storage,
preparation, for obtaining sufficiently high-quality genomic DNA for array CGH. DNA isextracted, stored and handled using appropriate conditions and methods.
(1) Inappropriate conditions unsuitable for testing
Detection sensitivity is remarkably diminished when a tumor tissue or cells arecontaminated with a substantial proportion of normal tissues or composed with aheterogeneous cell population. A part of normal tissues or cells need to be removed as muchas possible.
When the quality of the recovered DNA is low, the error of the examination results can besignificant.

20(2) Cause
The low ratio of tumor cells in tissue / cell samples is caused by insufficient removal of
the normal cells from the whole sample used for DNA extraction. The quality of therecovered DNA is influenced by the target of the sample, conditions of storage, sample
preparation, recovery and storage of extracted DNA.

(3) Troubleshooting
Remove normal cells by an appropriate procedure such as micro/macro dissection.
The abundance ratio of tumor cells can be confirmed in the samples by analysis including
cytology and pathological examination on a split part of the samples.
If the quality of the recovered DNA is judged to be inappropriate for array CGH analysis,
repeat the DNA extraction and re-examine. If the specimen itself is of poor quality, resample the specimen and re-examine.
(4) Measures to avoid inappropriate situations
A portion of the collected sample should be stored for reexamination. If the specimen itself
is of poor quality, perform reexamination from the re-sampling.

4. Next Generation Sequencing (NGS)
Quality of samples used for DNA or RNA analysis by next generation sequencing(hereinafter referred to as NGS) is assured by conforming to specimen type-specific collection,
storage, transportation and treatment.
When performing sequencing using NGS, appropriate procedures and quality assurance inall the steps are important, regarding (1) proper collection and storage of specimens, (2)quality control of DNA or RNA, and (3) processing for sequencing reaction (library
preparation). These qualities affect the final sequencing efficiency and accuracy.
Although general quality management in NGS analysis will be described below, the quality
control method can be different depending on the components of NGS system used foranalysis.
4.1 Sample Quality Control for NGS Analysis
4.1.1 Blood
When DNA or RNA derived from nucleated cells is used for NGS analysis, a specified
amount is collected using a blood collection tube containing a nucleic acid stabilizer or a bloodcollection tube containing an anti-coagulant (EDTA or the like). Immediately thereafter, thetube should be inverted and mixed, and DNA or RNA is prepared from the blood specimen
according to the recommended method of a reagent kit that can extract DNA or RNA.

21

When circulating free DNA (ccfDNA) is used for NGS analysis, blood samples are collected
into a blood collection tube containing an anti-coagulant (EDTA or the like) or a bloodcollection tube containing a reagent with cytoprotective action. DNA is prepared according to
the recommended method of a reagent kit which can separate plasma (or serum) underappropriate conditions and extract ccfDNA.
(1) Inappropriate conditions unsuitable for testing
Coagulated blood or blood after freezing and thawing is unsuitable. When ccfDNA isused for NGS analysis, specimens in which nucleated blood cells are lysed are also
inappropriate. Specimens that should not be used for DNA or RNA extraction can not be
visually distinguishable in some cases. For this reason, record the history of collection,
storage, and transportation specimens so that they can be tracked.
(2) Cause
Inadequate properties such as coagulation and lysis of nucleated blood cells are caused byinsufficient mixing after blood collection. In addition, it is considered that the quality of
DNA or RNA to be extracted depends on long-term exposure to high temperatures,
repeated cycles of freezing and thawing, and storage or transportation out of theappropriate conditions (temperature and period). The quality of DNA or RNA deteriorates
when the reagent kit for DNA or RNA extraction and the blood collection tube are stored
in an environment deviating from the recommended conditions, and when used in a method
other than the recommended method.
(3) Troubleshooting
If it is judged that the quality of the recovered DNA or RNA is not suitable for NGS
analysis, re-testing is performed from DNA or RNA extraction. If the specimen itself is ofpoor quality, re-collect the specimen.
(4) Measures to avoid inappropriate situations
Immediately invert and mix tubes after taking blood. Samples should be stored under theconditions suitable for DNA extraction shown below. Generally, when DNA derived from
nucleated cells is extracted, it is desirable to perform DNA extraction within 24 hours after
blood collection, and it is possible to store up to 3 days under refrigeration conditions. If
DNA cannot be immediately extracted, it can be stored frozen, but should not undergo
repeated cycles of freezing and thawing. When ccfDNA is extracted, it is recommended touse a blood collection tube containing a ccfDNA profile stabilizer (for details see section 7.1
Collection of blood circulation free nucleic acid (1)). The preservation method of the blood
specimen after the blood collection should comply with the conditions described in the
package insert of the blood collection tube. If a dedicated blood collection tube is not used,22

immediate refrigeration or plasma separation is necessary. On the other hand, refrigerated
specimens for RNA extraction can be stored up to 24 hours, but it is desirable to treat withappropriate RNase inhibitors as soon as possible after collection. The blood collection tube
and the reagent kit for DNA or RNA extraction should be selected to conform to the type of
specimen and the purpose of analysis, and appropriately stored and managed as describedin the accompanying instruction manual. Operate whole processes according to themethods prescribed.
4.1.2 Tissues / Cells
The RNA profile can change prior to stabilization by freezing of tissues by a variety of factors,
such as induction of gene expression/suppression of RNA degradation. The degree of influence
depends on temperature, length of time exposed to ischemia, and storage time in ambient
temperature. Warm ischemia time during surgery has a greater effect than cold ischemia
time after organ tissue removal. The degree of influence due to the length of the storage
period depends on the amplicon size and the position of the primer or probe. When using
fresh frozen tissue specimens for NGS analysis, cryopreservation in a condition as close as
possible to their in vivo status is ideal. To do this, it is important to recognize lesions correctlyin their fresh state before the surgical specimen is fixed with formalin or the like and tocollect the viable cell population of the lesion as quickly as possible. However, when collecting
tissue specimens for research, do not prevent pathological diagnosis, such as mistakenly
collecting the parts necessary for pathological diagnosis. Tissue samples for research should
be collected from residual samples that are not necessary for pathological diagnosis.
From the surgical specimen, collect tissue specimens for NGS analysis from suitablesampling sites and avoid compromising pathological diagnosis (samples shall be taken
without having patients at a disadvantage) by avoiding sampling from areas where nucleic
acid and proteins have been denatured such as bleeding lesions and areas of necrosis. For
example, in case of a cancer, specimens are taken from both the cancerous and the noncancerous part (appropriate control part). A minimum description about the collection site
shall be recorded. From the surgical specimen, it is desirable to collect tissue specimens for
NGS analysis promptly. If this is not possible, temporarily store the specimens in arefrigerator (4 °C) or the like and collect the tissue specimens stored at 4 °C within 3 hours

alternatively. When extracting DNA from tissues or cells for NGS analysis, freeze the tissues(pellets in the case of cells) or store them in a refrigerator or freezer after immersing in thedesignated preservation solution.
When the specimen is to be cryopreserved, the amount to collect depends on the judgmentof the pathologist or clinician who is familiar with the requirements of pathology diagnosis.

23However, when there is no particular obstacle to pathological diagnosis and an adequate
collecting site can be secured, it is appropriate to collect tissue about the size of the tip of thesmall finger (about 1 × 0.5 × 0.3 cm, about 50 to 100 mg). Cut the tissue into cubes with 2-3
mm sides. Place a piece of tissue into a screw-capped tube that is resistant to lowtemperatures, and immerse the tissue in a nucleic acid protective agent or place the tube
directly in liquid nitrogen. Quick freezing should be performed, within 30 minutes of removalof the surgical specimen. When storing for a long term, keep the samples in a liquid nitrogenstorage container (about -180 °C.) until they are used. It is also possible to preserve for a longterm in a deep freezer (-80 °C.).
In NGS analysis using FFPE specimens, the existing methods used in pathological diagnosis
work at each facility should be respected. On the other hand, care should be taken in
obtaining DNA or RNA suitable for NGS analysis as much as possible. When handling tissue
specimens for treatment purposes, follow the methods recommended or required in the"Guidelines on the handling of pathological tissue samples for genomic research andtreatment: Standard operating procedures based on empirical analyses" issued by theJapanese Society of Pathology.
After removing the specimen, immerse it in a fixative as soon as possible and fix it. Even if
fixation can not be performed immediately, it is desirable that the specimen be stored in arefrigerator (4 °C.) or the like and fixed within about 3 hours. Transport the specimen
promptly at 2-8 °C. It is preferable to use a neutral buffered formalin solution for fixation
instead of a non-buffered (acidic formalin) solution. Also, when mainly considering extractionof DNA and genetic mutation analysis, it is desirable to fix with 10% formalin (3.5%
formaldehyde) rather than 20% formalin (7% formaldehyde). Some commercially available
tissue fixing solutions that do not contain formalin can be used to enable histologicalobservation and have been confirmed to be superior in the preservation of nucleic acids,
proteins and the like. It is most desirable to avoid over-fixing and to process within 24 hours
of surgery. However, if processing takes place within 48 hours after surgery, a reasonably
good retention of nucleic acids and the like can be expected.
In general, it is recommended that nucleic acid extraction from unstained specimens be
carried out promptly after sectioning and if unstained specimens need to be preserved for anindefinite period of time for research, they should be stored at 4 °C. However, if it is not
extremely long term, storage at room temperature would not generally affect the quality of
the nucleic acid. In the case of analysis from sliced sections of cancer tissue, it is necessary
to recover as much of the tumorous part as possible, in order to reduce the influence of
background noise caused by a large amount of normal cell tissue. There are no strict

24restrictions on paraffin coating of unstained specimens. A paraffin coating on the surface of
unstained specimens can reduce dust and physical damage and does not generally affect thequality of nucleic acid. Exposure to extreme conditions such as direct sunlight should be
avoided when preserving unstained specimens for long periods of time. Where there is apossibility of subjecting a specimen containing hard tissue to genomic research, avoid rapid
decalcification using acid such as by the Plank-Rychlo method which has a strong effect on
the quality of nucleic acid. Instead, perform slow decalcification using EDTA.
When DNA is extracted from tissues and cells and NGS analysis is performed, only tissues(pellets in the case of cells) should be frozen. Alternatively, store tissue in a special
preservative solution and maintain in a refrigerated or frozen state.
The success of mutation analysis by NGS depends on DNA amount, dsDNA (doublestranded DNA) concentration, type of specimen, and cellularity of specimen. The amount of
DNA required for the analysis depends on the measurement method to be used, the numberof target sequences of interest to be detected, the method of separating or concentrating thetarget cells, and the method of preparing the library. In the case of puncture aspiration or
needle biopsies, the amount of DNA obtained tends to be small. In NGS analysis targetingcancer, panel tests using DNA or RNA have become widespread. For small tissue specimens
and cell specimens, a method of simultaneously extracting DNA and RNA is used as needed.
The proportion of tumor needs to be 10-20% or more (due to the possible influence of
inflammatory cells, blood, mucin, necrosis). The main contributors to poor quality such as
DNA degradation are the use of formalin-fixed specimens or decalcification of metastatic bone
cancer specimens using strong acid.
(1) Inappropriate conditions unsuitable for testing
In the case of tumor tissues or cells which are highly contaminated with normal tissues,
the detection sensitivity decreases remarkably and false-negative results are common. If
the quality of the recovered DNA or RNA is low, errors in the measurement results will
increase.
(2) Cause
In a specimen used for DNA or RNA extraction, the proportion of the tumor cells in thetissue specimen decreases when the normal tissue is not adequately removed. The
quality of the recovered extracted DNA or RNA is influenced by the storage condition of
the specimen, the DNA recovery method, the extracted DNA storage conditions, and its
handling.
(3) Troubleshooting
Confirm the ratio of tumor cells by cytology, and pathological examination. If necessary,25

remove normal cells by micro/macrodissection or the like.
If it is judged that the quality of the recovered DNA or RNA is not suitable for NGS
analysis, perform the re-examination by repeating the DNA or RNA extraction process. If
the specimen itself is of poor quality, collect a fresh specimen.
Efficiencies of adapter ligation and DNA amplification for sequencing could decrease
when using DNA derived from FFPE due to factors such as fragmentation, crosslinking
with protein, and a low proportion of single-stranded DNA. This may make it difficult toprepare libraries. There are reagents available to check the quality of DNA for NGS
analysis.
NGS FFPE QC kit (Agilent Technologies)
TruSeq FFPE DNA Library Prep QC Kit (Illumina)
KAPA hgDNA Quantification and QC Kit (Kapa Biosystems)
Fragmentation can also be high in RNA derived from FFPE. Because of this, efficiencies
in adapter ligation and PCR amplification can decrease, making it difficult to prepare
libraries. Since the quality [RIN (RNA Integrity Number) value] that can be handled foreach library preparation reagent is different, it is desirable to consider whether theacquired extracted RNA is suitable for analysis based on the information from the
manufacturer and distributor.
If the quality of RNA does not satisfy the recommendations of the reagents being used toprepare the library, defects such as misjudgment of the gene expression level andinaccuracy of the detection of fused genes can occur in some cases.
(4) Measures to avoid inappropriate situations
Divide the collected sample, and subject a part of it to cytology, pathological examinationand the like to confirm the relative ratio of tumor cells, and submit a sample that has been
confirmed to have tumor cells present. The normal cells should be removed from tissue by
microdissection or the like as necessary. If it is judged that the quality of the extracted
DNA or RNA is not suitable for NGS analysis, perform re-examination by repeating DNA
or RNA extraction. Extraction, storage and handling should be carried out underappropriate conditions and methods. Keep a portion of the collected sample for reexamination. In cases where there is only a small amount of specimen, such as aspiration
cytology, pay attention to the low amount and diminished quality of extracted nucleic acid.
Properly stored FFPE specimens that are up to 5 years old are considered suitable foranalysis. Avoid using FFPE specimens that have been stored for more than 10 years. If thespecimen itself is of poor quality, collect a fresh specimen.

26

4.2 DNA and RNA Quality Control for NGS Analysis
4.2.1 General Precautions on Preparation of DNA and RNA
In preparing DNA or RNA for NGS analysis, the following general points should be noted.
・When performing the procedure, to avoid contamination of nuclease into reagents, always
wear powder-free laboratory gloves, and use pipettes fitted with nuclease-free pipette tips
and aerosol prevention filters.
・The experimental area shall always be kept clean, and experiments are carried out underconditions that satisfy Biosafety Level 1 (BSL 1).
4.2.2 DNA Quality Control
Length, amount, chemical purity and structural integrity can be all used as indicators of
the quality of DNA.
(1) Length
Generally, electrophoresis is used to confirm the length (degree of degradation) of DNA.
For genomic DNA with a good quality and a low degree of degradation, high molecularweight DNA can clearly be confirmed, whereas low molecular smear bands and peaks are
not observed.
In the case of DNA extracted from a FFPE sample, it may be suspected that the nucleic
acid is low molecular weight (fragmented) due to formalin fixation. In evaluating thequality of such DNA, it is effective to adopt a method of estimating the degree of DNA
degradation by real-time PCR using a couple of primer sets designed to have PCR productof different lengths at the same site.
In the case of ccfDNA extracted from plasma or serum, the majority of fragments range
from 140 to 200 bp in length. When high molecular weight DNA is observed, it is suspected
to be due to contamination by genomic DNA from nucleated cells, so it is not suitable foranalysis of ccfDNA.
Since high molecular weight DNA can be fragmented by physical stress, attention isrequired such as gently tapping with a finger when mixing solutions. Avoid repeated cyclesof freezing and thawing as much as possible.
(2) Amount
Since the concentration of DNA affects the efficiency of library preparation, it is necessary
to accurately quantify only the appropriately long dsDNA. Incorporation of single-stranded
DNA, RNA, or nucleotides can compromise accurate quantification.
In the ultraviolet-visible spectrophotometer, substances having absorption near 260 nm
are quantitatively measured together, so there is a tendency to overestimate the amount27

of DNA. Therefore, it is desirable to quantify a sample using a fluorescence assay methodor the like showing selectivity for dsDNA. In this case, it is necessary to prepare acalibration curve at an appropriate frequency and measure the DNA of known
concentration every time to obtain the measurement value as accurate as possible.
When extracting ccfDNA from plasma or serum, generally 1 to 50 ng of ccfDNA can berecovered from 1 mL of plasma. Although there is a range depending on the state of thepathological condition or other factors, in the case of an excessive yield, it is suspected tobe due to contamination with genomic DNA from nucleated cells, and thus not suitable foranalysis of ccfDNA.
When frozen DNA stock solution is used for preparation of a library for NGS, it is desirable
to quantify immediately before use.
(3) Chemical purity
A good index of chemical purity of DNA is that the value of A260/A280 is 1.8 to 2.0 and thevalue of A260/A230 is greater than 1.0. In addition, when EDTA, ethanol, phenol or the likeis contained in a solvent dissolving the DNA, it can inhibit the reaction of the library
preparation reagent.
Refer to the instruction manual provided by the manufacturer and distributor and checkthat the buffer does not include substances that would contaminate your sample.
(4) Structural integrity
There is a method of calculating the proportion of dsDNA as an index of structural
integrity. DsDNA is quantitated using a fluorescence spectrophotometer and comparedwith the DNA concentration measured with a spectrophotometer.
Percentage of dsDNA (%) = dsDNA concentration / DNA concentration × 100
When DNA with a high degree of degradation such as derived from FFPE is used, it may
be effective to prepare a library by calculating the necessary amount of DNA based on the
dsDNA concentration.
4.2.3 RNA Quality Control
The quality of RNA is defined by indexes of length, amount, chemical purity and structural
integrity.
(1) Length
Generally, electrophoresis is used to confirm the length (size) of RNA. It is desirable touse an electrophoresis system that can analyze a trace amount for quality of RNA for NGS
analysis. For RNA with a good quality and a low degree of degradation, 28S and 18S bands
can be clearly confirmed, and low molecular smear bands and peaks are not observed.

28

(2) AmountIf the concentration of RNA is not accurately quantified due to DNA or nucleotide
contamination, library preparation efficiency can be reduced.
(3) Chemical purity
A good chemical purity of RNA is indicated by an A260/A280 value of 1.8 to 2.0 and an
A260/A230 value greater than 1.0. In addition, when EDTA, ethanol, phenol and the like arecontained in a solvent dissolving RNA, or DNA is mixed in a sample, the reaction of thelibrary preparation reagents can be inhibited in some cases. Refer to the instruction
manual provided by the manufacturer and distributor, and check that the buffer does notinclude substances which listed as contaminants in the manual.
(4) Structural integrity
Measure the RIN (RNA Integrity Number) value using electrophoretic method(Bioanalyzer, etc.) as an index of structural integrity. The closer the RIN value is to 10, thehigher the quality of the RNA.
4.3 Library Quality Control for NGS Analysis
For NGS analysis for research, whole genome sequencing may be performed in some cases.
However, when conducting NGS analysis from the aspect of genetic testing, in most cases
only specific genes and genomic regions are used as targets for sequencing. In order toefficiently extract the maximum sequence results from valuable specimens and accurately
evaluate genetic mutations, it is extremely important to properly perform the quality checks
at each step described below. A library preparation reagent for NGS analysis is commerciallyavailable from various manufacturers and distributors, and the quality check methods ateach step are described in detail in each protocol, which are to be referred to.
4.3.1 General Precautions on Library Preparation
In the preparation of DNA or RNA for NGS analysis, the following general points should be
noted:
・When performing the procedure, to avoid mixing with nuclease into reagents, always wear
powder-free laboratory gloves, and use pipettes fitted with nuclease-free pipette tips andaerosol prevention filters to avoid mixing with nuclease into reagents.
.
・Keep the measurement area shall be always clean, and measurements are carried out underconditions of Biosafety Level 1 (BSL 1).
・In order to avoid mutual contamination between DNA libraries for NGS, it is preferable to29perform the procedure before and after PCR in different laboratories, and also to use
dedicated equipment and instruments. Also, after completing library preparation work,
instruments, laboratory benches and other work surfaces should be cleaned with bleaching
agent (sodium hypochlorite).
4.3.2 Quality Check of Fragmented DNA
In order to analyze the target region, when enriching the region by applying hybridization
between the probe and the target region, genomic DNA is fragmented first.
In order to decrease the loss of genomic DNA as much as possible, it is desirable to use
equipment that intensively sonicates the sample with a high powered and stable ultrasonicfrequency. Alternatively, there are reagents that combine multiple restriction enzymes or use
DNA fragmentation enzymes that cut at random sites.
Fragmented DNA is confirmed by an electrophoretic histogram of a microelectrophoresis
apparatus to have a single peak, and also checked for a peak shape, a size of the peak top,
and a size of the peak tail, according to the instructions of the manufacturer or distributor.
The optimum fragment length differs depending on the platform and reagent of NGS used
for the analyses, and the purpose of them.
Agilent Technologies' HaloPlex provides dedicated Enrichment Control DNA (ECD) alongwith reagents to check if restriction enzyme treatment is proceeding as expected.
For the experiment, it is recommended that a quality check is performed by including thisECD for one specimen. ECD contains genomic DNA and 800bp PCR product with cleavage
sites for all restriction enzymes used. When there is no problem with restriction enzyme
digestion, smear bands derived from genomic DNA are observed between 100 and 2500bp.
Three major bands around 125, 225 and 450bp corresponding to restriction enzyme digestion
fragments derived from the 800bp PCR product should be also observed. If these three bands
are seen, restriction enzyme digestion is regarded as successful.
4.3.3 Confirmation of Success or Failure of Adapter ligation and Quality Check
In order to analyze the target region, when enriching the region by applying hybridization
between the probe and the target region, an adapter for the NGS reaction is added to bothends of the fragmented DNA.
If the amplification product is obtained as expected by PCR amplification using the adapter
sequence, it can be considered that the adapter addition reaction is successful.
In order to obtain a high capture efficiency in hybridization, it is important to accurately
use the specified amount of the DNA library to which the adapter is added.

30

The adapter-attached DNA library is analyzed by an electrophoretic histogram of a microelectrophoresis apparatus, confirmed to have a single peak, and checked for peak shape, size
length of peak top, size of peak tail and the like. The optimum fragment length differs
depending on the platform and reagent of NGS used for the analyses, and purpose of theanalyses. In addition, quantitation of the library concentration by quantitative PCR is
recommended. Since quantitative PCR can selectively quantify only the library to which theadapter is added at both ends, the accuracy of the quantitative value is high. Quantitation
can be also performed using a fluorescence assay (or the like) showing specificity for dsDNA.
In such quantification, one should obtain as accurate measurement values as possible bycreating standard curves at an appropriate frequency and measuring DNA of known
concentration each time.
4.3.4 Quality Check of DNA library for NGS
The quality of the DNA library for NGS is confirmed by electrophoretic histogram diagramof microelectrophoresis apparatus.
When preparing a sample by target enrichment using the hybridization principle, confirm
that it has a single peak, and check for the peak shape, a size of the peak top, a size of thepeak tail and the like. When preparing a library based on the amplicon principle, multiple
peaks can be detected in some cases. The optimum peak shape and fragment length will
differ depending on the platform and reagent of NGS used for analysis, purpose of analysis
and so on. In addition, quantitation of the library concentration by quantitative PCR is
recommended. Since quantitative PCR can selectively quantify only the library to which theadapter is added at both ends, the accuracy of the quantitative value is high. Quantitation
can be performed using a fluorescence assay or the like, specific to dsDNA. In such
quantification, one should obtain as accurate measurement values as possible by creating
standard curves at an appropriate frequency and measuring known concentration each
time.

31

5. Circulating Tumor Cell (CTC) Measurement (cancer diagnosis / peripheral blood /trace amount cells)
CTC is a beneficial tool for obtaining information on cancer metastasis. Since the numberof CTCs flowing in the blood is extremely small, it is necessary to be able to capture the CTC
existing as low as only one cell per 100,000 leukocytes. Extreme caution is required for the
specimen preparation. A variety of measurement methods are commercially available,
including the only equipment approved by the FDA, namely the Cell Search system. It is
important to capture a very small number of CTC without damages, measure the number of
captured cells, and if possible, analyze the captured cells for subsequent genetic testing.
A general sample processing method can be described as follows,
1） The necessary amount of blood (for example 5 to 20 mL) is collected from the patient andstored in an appropriate tube (e.g. Cell Save) for stable storage. The possible storage
period varies depending on the type of tube and the measuring method. The analyzing
process should be performed within the period instructed by the manufacturer. Whenthe collected blood sample is transported during this period, storage conditions(temperature, humidity) should be controlled in accordance with the conditions
instructed by the manufacturer.
2） Buffer solution provided in a kit and a density gradient separating reagent are added topart or all the blood collected in process 1) and mixed. The sample is centrifuged to obtainblood cell components including CTC. Red blood cells can be removed by thecentrifugation or hemolysis treatment.
CTCs are concentrated using various methods from the blood cell components after theprocess 2). It is important to consider that CTCs need to be recovered with a high yieldthrough the concentration process, and damage of CTC should be minimized. Processes
should comply with protocols provided by manufacturer and distributor of the CTC
measurement system.
(1) Inappropriate conditions unsuitable for testing
Samples with a low CTC recovery rate or with damaged CTC due to inappropriate
conditions during collection, transportation, storage, or concentration are unsuitable fortesting, as CTC-derived DNA will not be obtained or detected sufficiently.
(2) Cause
The number of CTCs in the specimen is extremely small, and they can be lost or damaged
during the collection process.
(3) Troubleshooting
Review the processes as instructed by the manufacturer and select appropriate procedure32

in those processes. Concentrate and recover cells with less stressful conditions.
(4) Measures to avoid inappropriate situations
Avoid any collection methods that stress the cells and adopt a measurement method thatallows observation of CTCs.

-----------------------------------------------------------------------------------------------------------------------CTC；Circulating Tumor Cell
"Circulating tumor cell" or "peripheral blood circulating tumor cell" is defined as cells released from a
primary tumor tissue or metastatic tumor tissue and leaked into blood.

33

6. miRNA · Exosome
6.1

microRNA（miRNA）

miRNA is a very small, highly conserved RNA molecule that has a crucial role in biologicalprocesses. Most RNA extraction methods are specifically provided to obtain only mRNA. It isdifficult to efficiently prepare short RNA molecules. For this reason, extraction reagents or
kits intended to prepare short RNA molecules are desirable.
Unless analysis occurs immediately after extracting miRNA from the collected specimen,
the specimen should be kept under appropriate conditions. The extracted miRNA should be
stored in RNase-free conditions at -80 °C in a storage container preventing RNA adsorption.
If body fluids such as serum and plasma are used as a specimen and miRNAs secreted from
cells are targeted, cell contamination and hemolysis shall be avoided. When plasma is used,
an anticoagulant such as EDTA or citric acid should be used. Anticoagulants of biologicalorigin such as heparin should be avoided.
In the case of using frozen sections, tissue pieces should be frozen in liquid nitrogen andstored in an airtight container at -70 °C or below.
The extraction method set for each test or sample type should be followed. The storage
conditions for the extracted RNA should be controlled as recommended.
(1) Inappropriate conditions unsuitable for testing1） Degradation of RNA and contamination with impurities other than RNA.
2） When the amount of RNA is extremely small, it may not be possible to measureaccurately.
(2) Cause
1） RNA is degraded by being unnecessarily exposed to ambient or high temperature.
2） RNA is degraded by an RNA degrading enzyme.
3） RNA is adsorbed by contamination with impurities, containers and instruments.
4） In transportation, storage and measurement, RNA is adsorbed during one of theprocedure steps.
(3) Troubleshooting
1） Storage conditions of RNA should be -70 °C or lower.
2） For RNA storage containers, use containers designed to prevent adsorption of nucleic
acids.
3） When transporting RNA, keep it cool below -70 °C or use dry ice packaging.
4） The containers for transport shall be of a shape that is expected to maintaintemperature such as Styrofoam and that do not collapse RNA-containing tubes.
5） When RNA is used, confirm the presence or absence of contamination by impurities34

as much as possible by electrophoresis or absorbance measurement, and purify if
impurities other than RNA are observed.
(4) Measures to avoid inappropriate situations
1） When RNA is to be extracted from tissues or cells, use RNA protectant until
extraction or keep it at -70 °C or lower. When storing at -70 °C or lower, RNAdegradation can still occur depending on the tissue or cell type, so it is preferable tovalidate that a specimen is fit for purpose and not excessively decomposed beforehand.
2） If the sample type from which RNA is extracted is blood-derived, blood that has chyleor has undergone hemolysis should not be used.
3） When RNA is being extracted from serum, the storage temperature of the specimen
should be -70 °C or lower, and RNA extraction should be completed within 7 days
from the start of storage.
4） When RNA is being extracted from serum, the cycle number of freezing and thawingof the serum should be less than 3 times.
5） At the time of extracting RNA, ensure operating procedures are being followed, suchas procedures and conditions specified by each extraction reagent.
6） When storing RNA, in order to avoid repeated cycles of freezing and thawing, use
freezer without a defrost function.
7） The personnel handling RNA shall wear masks and gloves after removing RNase in
the handling environment, in order to prevent RNA degradation by RNA degrading
enzymes.
6.2 Exosome
Methods for separating exosomes include ultracentrifugation, precipitation, filtration andother methods. Comply with each isolation method or recommended isolation method for eachsample type. Avoid adding anti-coagulants of biological origin such as heparin in blood
analysis. Comply with the recommended storage conditions for isolated exosomes. When
extracting nucleic acid or protein from the isolated exosome, comply with the establishedmethod.
(1) Inappropriate conditions unsuitable for testing1） Influence of impurities other than exosome affects the results.
2） When the amount of isolated exosome is extremely small, it can not be measured
accurately.
(2) Cause
1)

Contamination of impurities in transportation, storage and isolation operation steps.

35

2)

An isolation method inappropriate for the measurement method is selected.

3)

Exosome is adsorbed to storage container.

4)

Sample is damaged by repeated cycles of freezing and thawing.

(3) Troubleshooting
1)

Remove cells and debris before exosome isolation.

2)

After isolation of exosomes, resuspend the sample in phosphate buffer saline (PBS)or the like.

3)

Store exosomes at 4 °C or less.

4)

Use a container with low adsorption for storage of exosomes.

5)

When transporting exosomes, use a refrigerator below 4 °C or pack in dry ice.

6)

Transport container is expected to maintain temperature such as styrofoam, and it
should be of a shape that does not collapse tubes containing exosomes.

(4) Measures to avoid inappropriate situations
1)

When extracting nucleic acid or protein from exosomes, extract immediately after
exosome isolation.

2)

At the time of the extraction procedure, comply with the operation procedure, suchas procedures and conditions specified by each extraction reagent.

3)

Avoid repeated cycles of freezing and thawing when storing exosomes.

7. Blood Circulating Cell Free Nucleic Acid
7.1 Blood Circulating Cell Free Nucleic Acid: Tumor
The general procedure from blood collection to plasma separation and storage is shown. (See
4.1.1 Blood section for inappropriate conditions, cause, troubleshooting, and avoidance
methods)(1) Collection· Collect blood using a blood collection tube according to the package insert of the reagent or
the device to be used or the instructions of the product/service provider.
· The use of a blood collection tube containing ccfDNA profile stabilizer is recommended.
These types are currently marketed in Japan:
PAXgene ccfDNA blood collection tube (Japan Becton · Dickinson)
Blood collection tube for cell free DNA extraction (Roche・Diagnostics)· When a blood collection tube with stabilizer can not be used, it is expected that inactivationof endogenous DNase will be required, therefore a blood collection tube with EDTA should
be used than other anticoagulants.

36

·Immediately invert and mix the tube after collecting blood. Mix slowly to avoid DNA
release due to destruction of cells. Invert and mix in accordance with the description of thereagent to be used or the package insert of the device or the instructions of the service
provider.

(2) Transportation· Be careful not to expose the sample to ambient temperature for a long period before plasma
separation, in order to avoid blood cell destruction.
· Plasma separation is performed according to the instructions of diagnostic reagent
manufacturer, the equipment manufacturer or laboratory service provider. Also note therecommendations for storage conditions.
• For blood collection tubes with stabilizer, maintain the stability of the ccfDNA profile byfollowing the package insert or information provided. If there are instructions from theservice provider, follow these.
• When using a blood collection tube with EDTA, detrimental effects can be avoided if
processing is within 6 hours at 2-6 °C, depending on the measurement application. If there
is a package insert or instruction sheet, follow the instructions.
• Validate the time and temperature conditions as necessary.
• As the ccfDNA profile changes due to freezing, the primary sample (whole blood sample)
should not be frozen.
(3) Pretreatment of ccfDNA before specimen treatment• Perform processing according to the package insert of the reagent or the equipment, or the
instructions of the service provider. The following is a general procedure to use a bloodcollection tube with EDTA, if one is not described in the package insert or instructions.
• Centrifuge at 1,600-2,500 × g, 2-8 °C, for 10 minutes. In order to avoid contamination of
nucleic acids from leukocytes, pay attention not to disturb the interface between plasmaand cell layer, and then transfer it to another tube.

-----------------------------------------------------------------------------------------------------------------------ccfDNA; circulating cell-free DNA
Extracellular free DNA fragments called circulating cell-free DNA exists in the liquid layer of human blood. It isalso called cell-free DNA (cfDNA), or blood circulating free DNA. DNA derived from a cancer, and that has beenreleased into the blood is called circulating tumor DNA (ctDNA).

37

• Perform the second centrifugation. This should be carried out under the conditions of
14,000 -16,000 × g, 2-8 °C for 10 minutes.
・In the absence of a high speed centrifuge, the second centrifugation can take place at alower relative centrifugal acceleration, for example 3,000 - 5,000 × g, 2-8 °C, for 20 minutes.
(4) Storage• The preservation of plasma should be in accordance with the package insert of the reagent,or the instructions from the service provider. If there are no instructions, it can be stored for
up to 24 hours at 2-8 ℃. Longer term storage should be at or below -20 ℃. Validate thetemperature and duration of storage.
7.2 Noninvasive Prenatal Genetic Testing
A prenatal genetic test using pregnant women's peripheral blood (maternal blood) has beendeveloped and started to be used in Japan, following overseas usage. For this test, peripheral
blood is collected as a specimen and used for measurement. Because it minimizes physicalburden on the patient, it is called non-invasive prenatal genetic testing (NIPT).
Fetal-derived DNA fragments account for 3-10% of the total maternal blood ccf DNA.
Its length tends to be short (less than 300 bp), suggesting it arise from apoptosis rather thancell necrosis. There are various ways to detect fetal numerical chromosome abnormalities byanalyzing fetal-derived DNA present in maternal plasma. The shotgun method of sequencing
all DNA in plasma and the method of detecting specific sequence markers are analyticallyvalidated in clinical trials.
NIPT measures the relative number of specific autosomes (13, 18, 21) by quantifying both
fetal-derived free DNA and mother-derived DNA fragment present in maternal plasma, andthus determines the presence or absence of a numerical abnormality of a fetal chromosome.
NIPT is a screening test and non-definitive test. Besides the specific autosomal chromosomechange, now detection methods of the numerical abnormalities of sex chromosomes have beendeveloped and applied to a practical use in oversea countries.
The emergence of fetal-derived DNA fragments in maternal blood is observed from as early
as week 5 of pregnancy, or after 9 weeks as the latest. Therefore, the examination should bedone after 10 weeks gestation.
For sample collection, it is recommended to use a blood collection tube containing ccfDNA
profile stabilizer (for details see section 7.1 Collection of blood circulation free nucleic acid(1)). Note the gauge size of the blood collection needle to avoid the effects of cell destruction
due to excessive pressure. Procedures such as inverting and mixing immediately aftercollection should follow the method described in the package insert of the blood collection

38

tube.
NIPT in Japan is conducted in clinical laboratories which have been certified by the Japan
Medical Association as a clinical research facility. Since plasma separation and analysis
procedures are strictly defined and implemented according to the standard work protocol ineach clinical laboratory, from the viewpoint of quality control of NIPT, it is necessary toconsider collection and transport conditions of blood specimens. For this reason, concrete
methods for risk management such as transport means of specimens and prevention of
mistakes should be presented. In addition, this examination should be requested in a limited
facility capable of providing adequate genetic counseling.
(1) Inappropriate conditions unsuitable for testing
Coagulated blood. Samples that include lysed nucleated blood cells. Samples stored forinappropriate times and temperature after collection and transportation can deteriorate
in quality and reduce the reliability of measurement results.
(2) Cause
Mixing after blood collection is insufficient. Plasma separation is insufficient. Influenceof freezing of specimen and pressure. Inadequate use of blood collection tube, long-term
storage of sample after collection, transport time and conditions and storage temperature
are not appropriate.
(3) Troubleshooting
If measurement is difficult due to inappropriate nature, blood should be taken again.

-----------------------------------------------------------------------------------------------------------------------Relative centrifugal acceleration RCF (Relative Centrifugal Force): The so-called "centrifugal force" used forcentrifugal separation is referred to as relative gravitational acceleration because it is expressed as a ratio with thegravitational acceleration of the earth.

39

(4) Measures to avoid inappropriate situations
Choose an appropriate blood collection tube, collect blood, immediately invert thoroughly
and mix, and store and transport appropriately as described in the attached instruction
manual.
In general, blood collection tubes with EDTA are designed to be kept refrigerated after
blood collection, but some blood collection tubes dedicated to ccfDNA extraction are designed
to be stored at ambient temperature without refrigerating or freezing of blood specimens.
Set the storage temperature appropriately. It is recommended to transport in dedicatedshipping box where specimen temperature control is secure from collection until delivery.
When transportation exceeds a predetermined time due to holidays or delays, plasma
separation and freezing (-80 °C) is performed at the registered commercial laboratories inaccordance with the standard operating procedure document prescribed by the NIPT clinicallaboratory.

8. Mitochondrial DNA
Genetic analysis targeting mitochondrial DNA (mtDNA) is performed by several methods
including using a PCR-RFLP method for detecting specific point mutations, Southernblotting, and whole genome sequencing with next-generation sequencers.
Blood is collected using a blood collection tube containing an anticoagulant (e.g. EDTA), then
immediately mixed by inversion. When nucleic acid extraction is not performed on the day ofblood collection, store the sample refrigerated.
Cryopreservation is not contraindicated but better to avoid in some cases. It depends on thedownstream processing method. For example, although platelets do not contain nuclear DNA,
they do contain mtDNA. Thus, platelets may be used as a source for mitochondrial DNA
isolation. Freezing the blood will make centrifugation more difficult, due to hemolysis or
destruction of white blood cells.
Tissue samples are stored frozen (-70 ℃ or below) in a sterilized container. The maximum
storage period for frozen tissues is recommended to be less than 1 month at -80 °C or lessthan 1 week at -20 °C.
(1) Inappropriate conditions unsuitable for testing1) Blood is contaminated with impurities or clotted (platelets are absorbed in blood clots,
and mtDNA content in the supernatant is thought to be reduced).
2) Accurate measurements can not be possible with extremely small amounts of isolated
mtDNA. It is difficult to judge if the measurement result has been affected bycontamination.

40

mtDNA has a high copy number per cell and the risk of contamination is high.

(2) Cause
1) Impurities contaminate the sample during transportation, storage and isolation
processes.
2) Genomic DNA is contaminated in the isolation procedure. It is known that thesequences homologous to mtDNA called NUMT (nuclear mitochondrial DNA segment)
are interspersed in genomic DNA. Nuclear DNA contamination should be controlled
when analyzing mtDNA regions containing sequences homologous to NUMT.
(3) Troubleshooting
1) The containers used for collection, and prevention of acquiring the operator's mtDNA
should be checked. An isolation procedure with a high recovery rate of mtDNA should
be selected. The contamination of genomic DNA should be avoided as much as possible.
Pay attention to the PCR primer design for analyzing the samples prepared fromtissues or whole blood, where the genomic DNA contamination cannot be completely
avoided, and take into account the existence of NUMT.
(4) Measures to avoid inappropriate situations
1) Use a sterile, specialized container is used for storing the sample.
2) Use samples suitable for mtDNA extraction including platelets.
3) Use a reagent specifically designed for mtDNA extraction is used for the isolation
operation.
4) Select an extraction method that takes into consideration that mtDNA is circular.

41

（4）Appendix
3-1: Quality Control of DNA for Array CGH
In the case of detecting changes in genome copy number using CGH microarray, quality
control of DNA is important because the quality of DNA used will affect the noise level of thedata finally obtained and the accuracy of the analysis result.
In the quality control of DNA, length, amount, chemical purity and structural integrity canbe used as indices. For confirming the length (degree of degradation) of DNA, electrophoresis
is used. For good DNA with a low degree of degradation, high molecular weight DNA can beclearly confirmed, and smear bands and peaks of low molecular weight are not observed. It
is preferable that the DNA is free of carbohydrates, organic solvents, proteins, and RNAs.
Further, the index of chemical purity of good DNA is that the value of A 260 / A280 is 1.8 to 2.0
and the value of A260 / A230 is higher than 1.0. There is a method of calculating the ratio of
double stranded DNA (dsDNA) as an index of structural integrity. dsDNA is quantitatedusing a fluorescence spectrophotometer and compared with the DNA concentration measured
with an absorptiometer.
Percentage of dsDNA (%) = dsDNA concentration / DNA concentration × 100
The quality of DNA is very important as it affects the final analysis result. It is importantto prepare DNA that meets the quality recommended by each manufacturer.
【index】
Evaluate with purified DNA quality.
1)

A260/A280

≧1.5

2)

A260/A230

≧1.0

2)

Confirm that there is a band of 20kbp or longer by electrophoresis and thatfragmentation is not evident.

3)

For example, the amount of double-stranded DNA is specifically measured using afluorescent reagent or the like.

* The amount of DNA required depends on the array platform to be used.

3-2: Quality Check of CGH Microarray DNA Specimen: Agilent CGH / CGH + SNP Microarray
When detecting changes in the genome copy number using the CGH microarray, the qualityof the DNA to be used affects the noise level of the data to be finally obtained, that is, theaccuracy of the analysis result. Therefore, before and after labeling DNA quality control isimportant. Here, a direct labeling method by Agilent Technology's CGH microarray and DNA
fluorescent label (hereinafter referred to as labeled) reagent for CGH + SNP microarray,
SureTag DNA Labeling Kit, SureTag Complete DNA Labeling Kit is cited as an example.

42

With reference to these kits, we describe the method of DNA quality check for CGH
microarray, relevant for both the control DNA and the DNA to be analyzed.
Quality check of genomic DNA and labeled DNA for CGH and CGH + SNP microarrays(1) Notes on operation· To avoid nuclease contamination into reagents, always wear powder-free laboratory gloves
and use pipet tips with an appropriate solution, and pipet tips with nuclease-free aerosol
prevention filters.
· Clean the measurement (or work) space at all times.
· Water used for dilution / buffer solution shall be free of nucleases.
· Do not mix solutions containing genomic DNA using a Vortex Mixer. Mix the liquid bygently tapping the tube with your finger.
· Solutions containing genomic DNA should not be subjected to repeated cycles of freezing
and thawing.
· When using a frozen stock solution, follow these directions.
① Melt the dispensed solution as quickly as possible so as not to heat at ambient
temperature or higher.
② Lightly mix with a Vortex Mixer for a few seconds, centrifuge for 5 to 10 seconds in acentrifuge, and spin down the liquid adhering to the tube wall and lid.
③ If DNA is not completely dissolved, dissolve by heating at 37 ℃ for 30 minutes.
④ Store on ice or in a cooling block until use.
· Perform measurement under conditions of Biosafety Level 1 (BSL 1).
(2) Quality check of genomic DNA before labeling operation·Confirm the values of A260/A280 and A260/A230 using ultraviolet visible spectrophotometersuch as NanoDrop (Thermo Fisher Scientific) to confirm that high concentrations of salt,
protein, organic solvents are not mixed in with the DNA. It is desirable that the value ofA260/A280 is between 1.8 and 2.0, the value of A260/ A230 is higher than 1.0. It is also desirable
to confirm that there is no contaminant affecting absorbance at 260 nm by continuously
scanning the absorbance at 220-320 nm.
· To confirm that there is no contamination by a large amount of RNA in the DNA,quantification is carried out by both a fluorescence analysis method specific to doublestranded DNA and an ultraviolet-visible spectrophotometer.

Then, confirm that there isno significant difference between the two values. Since quantitative values obtained byultraviolet and visible spectrophotometer include those attributable to substances having
absorption in the vicinity of 260 nm such as RNA, dissociation of the quantitative values

43

of both methods suggests the possibility of RNA contamination. Contamination of a large
amount of RNA affects the labeling reaction. The fluorescence analysis method is describedin the following item. At the time of measurement, it is important to confirm that the DNA
is completely dissolved. If the measured concentration exceeds 350ng/μL, dilute it two-fold
and measure again.
· To confirm degradation of genomic DNA, agarose gel electrophoresis or fully automatedhigh throughput electrophoresis, TapeStation, with genome DNA kit is used. Degradationof genomic DNA not only affects the yield of labeled DNA and efficiency of labeling but also
hinders the process of performing random fragmentation with restriction enzymes.
· For quantitative determination of genomic DNA, it is recommended to use a fluorescenceanalysis method which is specific to double stranded DNA like Qubit Fluorometer.
Quantitative values obtained by ultraviolet-visible spectrophotometer have a risk oferroneous determination due to contaminants in DNA as also noted in the above item. In
CGH and CGH + SNP microarrays, the amount of DNA is within the recommended DNA
amount range depending on each microarray format. It is important that the amount of
the DNA to be analyzed is the same as that of the control DNA competitively hybridized tothe microarray. Incorrect quantification of DNA not only causes the DNA used for labelingto deviate from the recommended range, but also causes deviation between the amount ofcontrol DNA and the amount of DNA to be analyzed. As a result, the assay noise (Derivative
Log Ratio Spread (DLRSD) of the data rises, and the detection power of the copy number
change region decreases, so it is important to quantify as accurately as possible. Quant-IT
assay using Qubit, and PicoGreen reagent by Thermo Fisher Scientific Inc. are used as
fluorescence analysis methods. When fluorescence analysis methods are used, it ispreferable to prepare a calibration curve at an appropriate frequency and to measure a
DNA of known concentration every time so as to prevent a decrease in the quantitativevalue due to repeated measurement, in order to obtain a measurement value that is as
accurate as possible.
(3) Quality check of genomic DNA after restriction enzyme reaction
For DNA labeling procedures of CGH and CGH + SNP microarrays, genomic DNA is firstfragmented with Alu I and Rsa I restriction enzymes. Separate the DNA after the restriction
enzyme reaction using 0.8% agarose gel electrophoresis, confirm that most of the fragmented
DNA is in the range of 200 bp to 500 bp. Due to the low concentration of DNA, it is desirable
to use a combination of high sensitivity detection reagents such as SYBR Gold Nucleic Acid
Gel Stain (Thermo Fisher Scientific).
(4) DNA quality check after labeling44Labeled DNA is prepared by annealing random primers to both the control DNA fragmented
with the restriction enzyme and the DNA to be analyzed, and performing exo-Klenow
fragment enzymatic reaction in the presence of Cyanine 3-dUTP or Cyanine 5-dUTP,
respectively. In the protocol which omits the restriction enzyme reaction, fragmentation iscarried out by performing longer heating when annealing random primers to the genomic
DNA. The labeled DNA is isolated by column purification, adjusted to a predeterminedcapacity depending on each microarray format, and labeled DNA yield and fluorescent dye
uptake rate are measured with an ultraviolet visible spectrophotometer.
Measure A260, A550, A650 values, calculate the concentration of labeled DNA from A260, theconcentration of Cyanine 3 from A550, and the concentration of Cyanine 5 from A650. The
labeled DNA yield (μg) is calculated by multiplying the DNA concentration by the above
prepared predetermined volume. Fluorescent dye uptake rates are calculated by dividing theconcentrations of Cyanine 3 and Cyanine 5 by the concentration of labeled DNA (pmol/ μg),respectively.
Estimated yield is 3-6 μg when labeling with 200 ng of DNA or labeling with 8 x format, 813 μg in case of 500 ng, 9-16 μg in case of 1 μg. Estimates of fluorescent dye uptake rates(Cyanine 3 and Cyanine 5) are 15 to 50 pmol/ μg when labeling with 200 ng of DNA or labelingwith 8 × format, and 20-60 pmol/ μg for 500 ng, 20-60 pmol/ μg for 1 μg. When using a protocol
omitting the restriction enzyme reaction, the fluorescent dye uptake rate is 5 pmol / μg lower
than the above value.
If the yield of labeled DNA is lower than the above-mentioned target value, it may be lost
due to a failure in the column used for labeling DNA purification. If the column elution
solution obtained in the purification process of labeled DNA is preserved, purify from it again.
If the yield is still low, it is desirable to confirm the set temperature of the equipment used
for the labeling procedure and the quality of the genomic DNA before labeling and to carryout labeling again.
When the fluorescent dye uptake rate is lower than the above-mentioned reference value, it
is expected that some problems may have occurred in the labeling procedure such asdegradation of the dye. It is important to confirm the temperature setting of the equipmentused for the labeling procedure. It is desirable to check the quality of the genomic DNA before
labeling and re-label it. There is also the possibility of degradation of the labeling reagent
depending on repeated freezing and thawing of the fluorescent dye and the storage
temperature of various enzymes, so confirm this point again.
Since it affects the power with which copy number changes can be detected, it is extremely
important to properly perform quality checks at each of the above-mentioned steps for45

efficient analysis of a valuable DNA specimen.

4-1: Quality Check of NGS DNA Library: SureSelectQuality control of samples and prepared libraries is important since the quality of DNA
sample used will affect the final sequencing efficiency when performing DNA target
sequencing using NGS. Here, we describe the method of quality checking in NGS library
preparation taking Agilent Technologies' target enrichment reagent SureSelect as an
example.
Library quality check for DNA target capture sequence (exon sequence, target sequence)(1) Notes on procedure• To avoid nuclease contamination of reagents, always wear powder-free laboratory gloves
when using the procedure, use the correct solutions, and use a pipette fitted with a pipette
tip with nuclease-free aerosol prevention filter.
· The experiment area should be kept clean at all times.
· Avoid rigorous mixing of solutions containing genomic DNA with a Vortex Mixer, andinstead mix solutions by gently tapping.
· Avoid repetition cycles of freezing and thawing as much as possible for solutions containing
genomic DNA.
· When using a frozen stock solution, work with it as follows.
①

Make sure not to heat at ambient temperature or higher and melt the dispensed
solution as quickly as possible.

②

Briefly mix with a Vortex Mixer for a short time, centrifuge in a centrifuge for 5-10
seconds, and spin down the liquid adhering to the tube wall and lid.

③

Store on ice or in a cooling block until use.

· Perform procedures under conditions of Biosafety Level 1 (BSL 1).
(2) Quality check of genomic DNA· Check the ratio of A260/A280 using an ultraviolet visible spectrophotometer such as
NanoDrop. The ratio of A260/ A280 should be between 1.8 and 2.0.
· To confirm the presence or absence of degradation of genomic DNA, use agarose gelelectrophoresis or fully automated high throughput electrophoresis, such as the genomic
DNA kit of TapeStation. If the degradation of genomic DNA is ongoing, the amount ofrecoverable DNA decreases after fragmentation, or genomic DNA not fragmented to thedesired length can occur.
· For quantitative determination of genomic DNA, it is desirable to use a fluorescence assay

46

method which shows selectivity for double-stranded DNA. When quantitating with anultraviolet-visible spectrophotometer, substances having absorption near 260 nm arequantitatively measured together, so there is a tendency to estimate the quantitative value
excessively. When the quantitative value is overestimated, fragmentation becomes uneven,the yield at purification deteriorates, and unexpected by-products are formed. Conversely, if
the quantitative value is underestimated, there may be cases where a library of the amountrequired for hybridization for target capture can’t be obtained. Quant-IT assay using Qubit
and PicoGreen reagent by Thermo Fisher Scientific Inc. are used as a fluorescence assay
method. When using the fluorescence assay method, in order to prevent lowering of thequantitative value by repeated measurements, it is preferable to prepare a calibration curveat an appropriate frequency and measure the DNA of known concentration each time toobtain an accurate measured value.
(3) Quality check of genomic DNA after fragmentation
In library preparation of NGS, genomic DNA is first fragmented in order to obtain an insert
size of a certain length according to the purpose of the sequencing.
In order to obtain DNA fragments of the desired length with as little loss as possible, Agilent
Technologies' SureSelect's protocol specifies the use of a Covaris ultrasonicator (M&S
Instruments) which uses Adaptive Focused Acoustics (AFA) technology that intensively
sonicates a sample with high power and stable ultrasound.
For DNA fragmented to the target length in the Covaris ultrasonciator, confirm the size ofthe fragmentation and confirm the quantitative value of fragmentation with a microchip typeelectrophoresis system Bioanalyzer or TapeStation (Agilent Technologies). As an example,
when performing all exon analysis with Illumina's NGS, the target peak size offragmentation is 150-200 bp. It should be a single peak with a peak top between 150-200 bp
in the electrophoretic diagram of the Bioanalyzer or TapeStation and with the same peak
shape as the figure described in the protocol. It is also desirable to measure yields. If a yieldremarkably far from the expected initial starting amount is obtained, it is necessary to startover from the quantification of genomic DNA.
(4) Quality check of DNA library with adapter after adapter addition, PCR and purification
DNA fragmented with the Covaris ultrasonicator goes through the steps of end repair, Atail addition, adapter addition, PCR amplification, bead purification, and hybridization tocapture and concentrate the sequence of interest. In this case, it is important to quantify the
DNA library added by the adapter and perform hybridization in the amount specified in the
protocol in order to obtain a high capture efficiency.
In the SureSelect protocol, use a Bioanalyzer or TapeStation for size confirmation and47

quantification. As an example, when exon analysis is performed with Illumina's NGS, theDNA library peak size after adapter addition should be about 250 bp-275 bp. It should be asingle peak with a peak top between 250 and 275 bp in the electrophoretic diagram of the
Bioanalyzer or TapeStation. Then, confirm that there are no other unnecessary peaks andshoulder, and that the peak observed has the same peak shape as the figure described in the
protocol. Further, the obtained peak is to be quantified. For whole exon analysis, a DNA
library with adapter of 750 ng is required for hybridization.
(5) Quality check of DNA library with adapter after capture, concentration and amplification
By hybridization with a biotinylated RNA probe designed in the target region of interest,
the captured adapter-attached DNA library is concentrated by streptavidin beads. Then,through the process of PCR and bead purification, which adds an index bar code for analyzing
multiple samples in one lane, it becomes a library for final sequencing with NGS. Since thisfinal library has a low concentration, a high-sensitivity DNA kit of a Bioanalyzer or
TapeStation is used for size confirmation and quantification. As high sensitivity kits are also
sensitive to contamination, day-to-day maintenance of equipment used, such as electrodewashing, is important for obtaining stable results. It should yield a single peak with a peak
top between 300-400bp in the electrophoretic diagram of the bioanalyzer or TapeStation.
Confirm that there is no PCR nonspecific amplification product and that it has a peak shaperesembling the figure described in the protocol. Typical yields (concentrations) are between
0.7 ng/μL and 1.2 ng/μL when whole exon analysis is performed with Illumina's NGS.
In addition, when adding a barcode to each sample and multiplexing multiple samples in
one lane and performing sequencing, it is recommended to quantify the library concentration
by quantitative PCR. Since quantitative PCR can selectively quantify only the library towhich the adapter is added at both ends, the accuracy of the quantitative value is high. A kit
for quantification of NGS libraries from Agilent Technologies Inc., Illumina Inc., and Thermo
Fisher Scientific Inc. can be used.
The target sequence by NGS is an analytical method which still requires considerable cost
and time, though running costs have drastically decreased in recent years. In order toefficiently obtain the maximum sequence results from valuable DNA specimens, it isextremely important to properly perform the quality checks at each of the above-mentioned
steps. In preparing DNA libraries for each NGS manufacturer, the quality checking method
at each step is detailed in each protocol, so refer to it.

4-2: Quality Check of NGS DNA Library: HaloPlex
Quality control of specimens and prepared libraries is important since the quality of DNA

48

specimens used will affect the final sequencing efficiency when performing DNA target
sequencing using NGS. Here, we describe the method of quality checking in NGS library
preparation taking Agilent Technologies' target enrichment reagent HaloPlex as an example.
【Library quality check for DNA target capture sequencing】
Notes on procedure and quality check of genomic DNA are basically the same as SureSelect(for details see appendix 4-1 NGS DNA library quality check section).
(1) About ECD control
HaloPlex is a method to create an NGS sequence library by capturing arbitrary sequencesof human targets of 5 Mb or less in total with high selectivity and efficiency by combining
hybridization, ligation and PCR techniques. It has several features such as the required
amount of DNA can be as small as 225 ng, the ability to amplify more than 200,000 amplicons
in a single tube, easy change of the target region, and a short experiment time. It is expected
to be used in desktop type next-generation sequencers such as Illumina's MiSeq.
In HaloPlex, restriction enzymes are used to fragment genomic DNA, so no equipment forphysical fragmentation like the Covaris ultrasonicator is needed. Dedicated Enrichment
Control DNA (ECD) is supplied with reagents as whether this restriction enzyme treatment
progresses as expected, being an important point in quality control. For the experiment, it isrecommended that quality control is performed by including this ECD for one specimen.
(2) Confirmation of restriction enzyme treatment
HaloPlex has been devised to improve the accuracy of mutation calls by covering the target
region with multiple amplicons, and genomic DNA is treated with 8 kinds of 16 restriction
enzymes determined. Whether 8 pairs of restriction enzyme treatment have been performed
without problems is confirmed by analyzing ECD samples treated with restriction enzymesof each set and undigested ECD samples with TapeStation or Bioanalyzer.
The ECD sample contains genomic DNA and a PCR product of 800 bp, and this PCR productcontains the cleavage sites of all restriction enzymes used in the HaloPlex kit. In undigested
DNA control, a band of genomic DNA larger than 2.5 kbp and a band of PCR product of 800
bp are observed. In restricted enzyme digested ECD samples, smear bands derived fromgenomic DNA are observed between 100 and 2500 bp, and three major overlapping bands
around 125, 225, and 450 bp. These three bands correspond to restriction enzyme digestion
fragments derived from the 800 bp PCR product, and the exact size after cleavage with 8
different RE master mixes are different. Minor bands other than the three major bands ofabout 125, 225, and 450 bp can be detected in the lane of the restriction enzyme digested
ECD sample. If three major bands are found, the defined enzymatic digestion can be49

considered successful.
(3) Quality check of final amplified DNA library
Restriction enzyme-treated DNA fragments are hybridized with a HaloPlex probe designed
for the target region. By hybridization with a HaloPlex probe, the target DNA fragment forms
a cyclic structure. Only the target DNA having the digested fragments of restriction enzymes
at both ends is ligated to form a cyclic structure. This target circular DNA is eluted, PCR iscarried out, bead purification is carried out, and the final library for the sequence iscompleted. In addition to hybridization, only the defined restriction enzyme-treated fragment
is ligated, and only DNA having a cyclic structure is PCR amplified, enabling capture to be
performed with extremely high selectivity.
Confirm and quantify the library size using the Bioanalyzer or TapeStation for thecompleted final library. In the example using Illumina's NGS device, the amplicon consistsof a 50 to 500 bp target DNA insert and a 125 bp sequencing motif. As a result, the ampliconsize ranges from 175 bp to 625 bp, confirming that most amplicons are distributed in therange of 225 bp to 525 bp. Use amplicons between 175 bp and 625 bp for quantification. Evenif peaks with sizes smaller than 175 bp are observed, they are not included in the subject ofquantification.
HaloPlex has also been reported to be applied to FFPE samples, and its flexibility and highcapture efficiency is expected to make it possible for new applications to be obtained by rapid
and comprehensive analysis of tens to hundreds or thousands of exon sites of target genes.
In order to stably bring out more reliable results, the quality check in these experiment steps
is very important.

4-3: Quality Check of NGS DNA Library: Ion AmpliSeq ™
When performing target sequencing using NGS, the quality of the DNA used will affect the
final sequence efficiency and the quality of the sequence. For this reason, quality control of
specimens is very important. Here, we describe the quality checking method in NGS library
preparation using multiplex PCR, taking Ion AmpliSeq ™ as a target enrichment method
provided by Thermo Fisher Scientific Inc., as an example.
Ion AmpliSeq ™ is a target enrichment method using multiplex PCR, enabling thesimultaneous amplification of up to 6,144 primer pairs from 1 to 100 ng of genomic DNA in
one tube. By adjusting the length of the amplification product at the primer designing stage,
it is also possible to deal with FFPE samples in which decomposition of nucleic acids occurs
more frequently.
(1) Notes on procedure50• To avoid nuclease contamination into reagents, always wear powder-free laboratory gloves,
use an appropriate solution, and use pipettes fitted with pipette tips with nuclease-freeaerosol prevention filters.
· The experiment area shall always be kept clean.
· Avoid rigorous mixing of solutions containing genomic DNA with a Vortex Mixer. Mix theliquid by gently tapping with your finger.
· Avoid repetition cycles of freezing and thawing as much as possible for solutions containing
genomic DNA.
· When using a frozen stock solution, work with it as follows:① Make sure not to heat at ambient temperature or higher and melt the dispensed
solution as quickly as possible.
② Briefly mix with a Vortex Mixer for a short time, centrifuge for 5 to 10 seconds in acentrifuge, and remove the liquid adhering to the tube wall and lid.
③ Store on ice or in a cooling block until use.
· Perform procedure under conditions of Biosafety Level 1 (BSL 1).
(2) Quality check of genomic DNA· Check the ratio of A260/A280 using an ultraviolet visible spectrophotometer such as
NanoDrop. The ratio of A260/A280 should be between 1.8 and 2.0.
· Measurement with an ultraviolet-visible spectrophotometer tends to overestimate theamount of DNA because the substance is quantitatively measured in the vicinity of 260 nm.
When the quantitative value is excessively estimated, there is a possibility that there will
be insufficient amplification products to make a library due to insufficient amount ofgenomic DNA as a template for PCR. For this reason, it is desirable to use quantitative
determination of genomic DNA by using a fluorescence assay method showing selectivity
for double-stranded DNA. As the fluorescence assay method, Qubit ™ dsDNA HS Assay
Kit using Qubit ™ and PicoGreen from Thermo Fisher Scientific Inc. are used. When using
the fluorescence assay method, in order to prevent the quantitative value from decreasing
due to repeated measurements, it is recommended to prepare a calibration curve at an
appropriate frequency and to measure the DNA of known concentration each time to obtainas accurate measured value. Otherwise, since 1 to 100 ng of genomic DNA corresponds to
300 to 30,000 copies, it is also possible to quantify the amount of DNA to be used byquantitating the copy number using TaqMan® RNase P Detection Reagent Kit (Thermo
Fisher Scientific) using real time PCR.
· Use genomic DNA kit of agarose gel electrophoresis or microchip electrophoresis to confirm
the presence or absence of degradation of genomic DNA. If the genomic DNA is degraded,51

amplification products enough for a library can not be obtained due to insufficient amountof genomic DNA as a template for PCR.
· In particular, it is important to confirm the decomposition degree of nucleic acids of FFPE
sample where decomposition of nucleic acid is seen more frequently and the yield of nucleic
acid is expected to be so small that it can not be analyzed by electrophoresis. For this reason,
it is desirable to adopt a method of estimating the extent of DNA degradation by real-time
PCR using a plurality of primer sets designed to have different lengths at the same site.
(3) Quality check of amplified DNA library
In the Ion AmpliSeq ™ technology, in order to sequence the target region, an adaptor foremulsion PCR and sequencing reaction is added after amplification of the target region bymultiplex PCR.
The success or failure of multiplex PCR and adapter addition reaction is judged by whether
enough adapter-added library DNA has been amplified.
For confirmation of the amount of this library DNA, use the Agilent ™ High Sensitivity
DNA Kit of Microchip type electrophoresis (Bioanalyzer ™) and calculate from the obtained
peak area, or by the Ion Universal Library Quantitation Kit of real time PCR. Quantitationof this library DNA affects subsequent amplification by emulsion PCR, so it is necessary to
perform it as precisely as possible.
However, in the case of homogenizing the amount of library DNA by the Ion Library
Equalizer ™ kit using the magnetic bead method, this process can be omitted.
The success or failure of the reaction of the library DNA with this Ion Library Equalizer ™
kit is judged based on the sequence result.
As described above, success or failure of preparation of library DNA by Ion AmpliSeq ™
depends largely on the quality of genomic DNA. Therefore, the quality check of genomic DNA
is important for stable, reliable results.

4-4: Quality Check of NGS RNA Library: TruSeq RNA Access
When sequencing RNA using NGS, the quality control of the specimen is important because
the decomposition degree and the amount of the RNA specimen used affect the final
sequencing efficiency. Here, Illumina's target enrichment reagent and TruSeq RNA Accessare taken as examples, and a method of quality checking in NGS library preparation withRNA as input is described.
【Library quality check for RNA target capture sequencing (target sequence)】(1) Notes on procedure52• To avoid nuclease contamination into reagents, always wear powder-free laboratory gloves
and use an appropriate solution, and a micropipette fitted with pipette tips with nucleasefree aerosol prevention filters when operating.
· The experimental area shall be kept clean and cleaning with RNase remover is also
performed.
· Avoid rigorous mixing of solutions containing RNA with a Vortex Mixer. Mix the liquid bygently tapping the tube.
· Avoid repetition cycles of freezing and thawing as much as possible in solution containing
RNA. Store at -80 ° C.
· When using a frozen stock solution, work with it as follows.
①

Melt on ice.

②

Briefly mix with tapping, and centrifuge for 5 to 10 seconds with a centrifuge, tospin down the liquid adhering to the tube wall and lid.

③ Store on ice or in a cooling block until use.
· Experiments are carried out under conditions of Biosafety Level 1 (BSL 1).
(2) RNA quality check· Check the ratio of A260/A280 using an ultraviolet visible spectrophotometer such as
NanoDrop. It is desirable that the ratio of A260/A280 is about 2.0.
· Use RNA kit of microchip type electrophoresis (e.g. Agilent TapeStation, Bioanalyzer) to
confirm degradation of RNA. Apart from the RIN value, the degree of degradation isevaluated by the ratio (DV 200: percentage of RNA fragments > 200 nt fragment
distribution value) of RNA fragments leaving a length of 200 bases or more. If RNA
degradation is severe, increase the amount of RNA used and make adjustments to ensurethat the library preparation is reliable. By these adjustments, decomposed RNA with RIN
value of about 2.0 can be flexibly used for library preparation.
・ For RNA quantification, it is desirable to use a fluorescence assay that shows selectivity
for RNA. When quantitating with an ultraviolet-visible spectrophotometer, substanceshaving absorption near 260 nm are quantitatively measured together, so there is atendency to overestimate amounts of RNA. When the amount is overestimated, there areproblems such as poor library yield, biased gene expression profiles, and unexpected byproducts. Quant-IT assay using Qubit and RiboGreen reagent by Thermo Fisher ScientificInc. are used as the fluorescence assay method. When a fluorescence assay is used, it isnecessary to prevent lowering of the quantitative value by repeated measurements. For
that purpose, prepare a calibration curve at an appropriate frequency and measure theRNA of known concentration every time to obtain a measurement value that is as accurate53

as possible.
(3) Quality check of library with adapter after addition of adapter, PCR, and purification
RNA goes through steps of heat fragmentation, reverse transcription, A-tail addition,
adapter addition, PCR amplification, bead purification, and then hybridization step tocapture and concentrate the sequence of interest. The amount of RNA and fragmentation
time should be adjusted according to the degree of degradation. In order to obtain highcapture efficiency, it is an important point as well as the reagent targeted for DNA to quantify
the library added the adapter and perform hybridization in the amount specified in the
protocol.
In the TruSeq RNA Access protocol, use a Bioanalyzer or TapeStation for size confirmationand quantification. The library peak size after adapter addition is about 250 bp - 275 bp. In
the electrophoretic diagram of the Bioanalyzer or TapeStation, confirm that there is a single
peak, with the expected peak shape, size length of the peak top, and size at the peak tail. For
quantification at this point, a fluorescence assay method or the like which shows selectivity
to dsDNA can also be used.
Prior to capture, up to 4 samples can be pooled at this stage and work efficiency can be
improved. In the case of pooling, it is desirable to refer to the degree of degradation of RNA
measured prior to library preparation and to mix specimens with close decomposition degrees.
The preparation of the library after capture is the same as that of SureSelect (For details,
see appendix 4-1 NGS DNA library quality check section).
TruSeq RNA Access is designed on the premise of application to FFPE samples. Due to its
high flexibility, applications such as comprehensive gene expression analysis and fusion genedetection are expected even for highly degraded RNA specimens. Even when RNA with highdegree of degradation is used, bias in the gene expression profile is unlikely to occur, enabling
more useful results to be produced. In order to stably bring out more reliable results, thequality check in these experimental steps is very important.

54

（5）Bibliography
1. Literature1） Dubbink HJ, Deans ZC, Tops BB, et al. Next generation diagnostic molecular pathology: criticalappraisal of quality assurance in Europe. Mol Oncol. 2014; 8: 830-9.
2） Chang F, Li MM. Clinical application of amplicon-based next-generation sequencing in cancer.
Cancer Genet. 2013; 206: 413-9.
3） Hagemann IS, Cottrell CE, Lockwood CM. Design of targeted, capture-based, next generation
sequencing tests for precision cancer therapy. Cancer Genet. 2013; 206: 420-31.
4） Aftimos PG, Barthelemy P, Awada A. Molecular biology in medical oncology: diagnosis,
prognosis, and precision medicine. Discov Med. 2014; 17: 81-91.
5） Collins FS, Varmus H. A new initiative on precision medicine. N Engl J Med. 2015; 372: 793-5.
6） Garraway LA, Verweij J, Ballman KV. Precision oncology: an overview. J Clin Oncol. 2013; 31:
1803-5.
7） Li T, Kung HJ, Mack PC, Gandara DR. Genotyping and genomic profiling of non-small-celllung cancer: implications for current and future therapies. J Clin Oncol. 2013; 31: 1039-49.
8） Reddington K, Tuite N, Minogue E, et al. A current overview of commercially available nucleic
acid diagnostics approaches to detect and identify human gastroenteritis pathogens.
Biomolecul Detect Quantificat. 2014; 1: 3–7.
9） Organization for Economic Cooperation and Development. Guidelines for Quality Assurancein Molecular Genetic Testing. 2007< http://www.oecd.org/dataoecd/43/6/38839788.pdf>
10） Gargis AS, Kalman L, Berry MW et al. Assuring the quality of next-generation sequencing inclinical laboratory practice. Nat Biotechnol. 2012; 30: 1033-6.
11） Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ Laboratory Standards for
Next- Generation Sequencing Clinical Tests. Arch Pathol Lab Med. 2015; 139: 481-93.
12） Chiu RWK, Chan KCA, Gao Y, et al: Noninvasive prenatal diagnosis of fetal chromosomal
aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl
Acad Sci USA. 2008; 105: 20458-63.
13） Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma
to detect Down syndrome: an international clinical validation study. Genet Med. 2011; 13: 913–
920.
14） Barrett AN, Zimmermann BG, Wang D, et al. Implementing prenatal diagnosis based on cellfree fetal DNA: Accurate identification of factors affecting fetal DNA yield. PLoS ONE. 2011;
6:e25202
15） Kosaka Nobuyoshi Editor, Circulating MicroRNAs: Methods and Protocols（2013）5516） Plebani M. The detection and prevention of errors in laboratory medicine. Ann Clin Biochem.
2010; 47: 101–110.
17） Hawkins R. Managing the pre- and post-analytical phases of the total testing process. Ann Lab
Med. 2012; 32: 5–16.
18） International Organization for Standardization: ISO 15189 （3rd. Ed.）. Medical laboratories-- Requirements for quality and competence. English-Japanese version. Japan Standards
Association. Tokyo. 2013, 1-50.
19） ISO/TS 20658 Medical laboratories examinations -- Requirements for collection, transport,
receipt,and handling of samples.
<http://www.iso.org/iso/catalogue_detail.htm?csnumber=68763>
20） Standardisation and improvement of generic pre-analytical tools and procedures for in-vitro
diagnostics. <http://www.spidia.eu/>

2. Guidelines etc.

56

The Member Lists of the Technical Committee onStandardization for Gene-based Testing and its Working Group(As of January, 2017)
The Technical Committee on Standardization for Gene-based Testing
Chairperson

Hayato Miyachi Professor of clinical laboratory studies based on basic medical
examination, Tokai University School of Medicine

Vice-chairperson Masayoshi Tsutsumi SRL Inc.
Member

Kiyoaki Watanabe Tokyo Clinical Diagnostic Medicine Center

MemberKunihiro Ueda Kobe Tokiwa University President

MemberTadashi Kawai International Clinical Pathology Center

MemberKiyoshi Kitamura University of Tokyo Professor of International Research Center ofMedical Education

MemberShinji Kosugi Professor, Graduate School of Medicine, Kyoto University

MemberFumio Takada Professor, Kitasato University Graduate School of Medical Sciences

MemberKenjiro Kosaki Associate Professor, Division of Medical Genetics Department ofPediatrics, Keio University School of Medicine

MemberToshihisa Ishikawa President, NGO Personalized Medicine & Healthcare

MemberMasato Maekawa Professor, Laboratory Medicine, Hamamatsu University School of
Medicine

MemberAkira Haneda Professor, Department of Public Health, Chiba University Graduate
School of Medicine

Member

Sakae Itoga Deputy Engineer, Chiba University Medical School Attached Hospital

MemberYoshinao Oda Professor, Department of Anatomic Pathology, Graduate School of
Medical Sciences Kyushu University

MemberShinobu Masuda Professor, Division of Oncologic Pathology, Nihon University Schoolof Medicine

MemberTomohiro Nakayama Professor, Division of Laboratory Medicine, Nihon University
School of Medicine

MemberNaohiro Noda National Institute of Advanced Industrial Science and Technology

MemberHiroki Nakae NPO Japan bio Measurement & Analysis Consortium

MemberKoichi Yamagata Sysmex Corporation

MemberYoshiaki Tazawa Roche Diagnostics Corporation57

Observer

Hiroshi Yoshikura Honorary staff of the former National Institute of Infectious
Diseases

ObserverYumako Yoshida Regional Medical Care Planning Division, Health Policy Bureau,
MHLW

ObserverMoe Arita Bio-Industry Division, Commerce and Information Policy Bureau, METI

ObserverYuhei Fukuda Bio-Industry Division, Commerce and Information Policy Bureau,
METI

The Working Group
Chairperson

Hayato Miyachi* Professor of clinical laboratory studies based on basic medical
examination, Tokai University School of Medicine

Vice-chairperson Masayoshi Tsutsumi* SRL Inc.
MemberMayu Takeda* Gifu University of Medical Science

MemberYutaka Hatanaka* Hokkaido University Hospital

Member

Sakae Itoga Chiba University School of Medicine affiliated hospital

MemberHiroki Nakae* Biochip Consortium for Specified Nonprofit Organizations

MemberNaohiro Noda National Institute of Advanced Industrial Science and Technology

MemberMitsuharu Hirai Arkray Co., Ltd.

Member

Hideaki Tazoe Arkray Co., Ltd.

MemberYasuhiro Izumisawa* Abbott Japan Co., Ltd.

MemberYasutaka Yuki Eiken Chemical Co., Ltd.

MemberSatoko Nakajo* SRL Inc.

MemberYoshiyasu Ogawa* LSI Mediation Co., Ltd.

MemberDaisuke Koga Otsuka Pharmaceutical Co., Ltd.

MemberKumiko Nojima** Sakura Finetek Japan Co., Ltd.

MemberYoko Hano Sysmex Corporation

MemberMasashi Hamamura* Sysmex Corporation

MemberKazuhiro Suzuki** Shimadzu Corporation

Member

Rie Takeda Sekisui Medical Co., Ltd.

MemberTomoyoshi Shiroshita Sekisui Medical Co., Ltd.

MemberAtsuo Mori** Nippon Gene Co., Ltd.

MemberMakoto Nagano* BML Inc., Ltd.

MemberKazumi Kenmochi BML Inc., Ltd.

MemberTadachika Yamazaki BML Inc., Ltd.

MemberAkio Matsushita Fuso Pharmaceutical Co., Ltd.

58

Member

Kazumi Sawakami Precision System Science Co., Ltd.

MemberNaoto Sawada* Ho Logic Japan KK

MemberNoriyuki Okita* Roche Diagnostics Corporation

MemberToru Ogawa* Roche Diagnostics Corporation

MemberKazuo Sakai** RIKEN Genesis Corporation

ObserverYuji Sekiguchi National Institute of Advanced Industrial Science and Technology

Observer

Masaki Sato Eiken Chemical Co., Ltd.

ObserverKazuhiro Okano* QIAGEN Corporation

ObserverMasayuki Ishikura** QIAGEN Corporation

ObserverTaro Ito** Sakura Finetech Japan Co., Ltd.

ObserverKatsura Uchida ** Sekisui Medical Co., Ltd.

ObserverMasamitsu Shikata Shimadzu Corporation* Document authors

Secretariat** Document review collaborators

Hideo Kato Director General, Japan Non-Profit Organization Japan Laboratory
Standards Council

Acknowledgment
A part of this research was supported by AMED under Grant Number
JP19kk0305005s0103.

59