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New Guidelines for NGS from the College of American Pathologists
Gerald Higgins
Posted: Thursday, August 2, 2012 5:02 AM
Joined: 3/7/2012
Posts: 2


They are very interesting. They will be used as a standard by the FDA and CLIA. I have posted some of them here:


 

 

 Sequencing 

Inspector Instructions: 

  • Sampling of sequencing policies and procedures
  • How does your laboratory ensure individual nucleotides are visualized adequately?
  • How does your laboratory interpret sequence variation?

 

MOL.34914         Gene Information          

Phase II 

There is adequate information about the gene being tested regarding its wild type sequence and reported mutations and polymorphisms. 

  

NOTE: DNA sequencing assays should be reserved for those disease genes that have been adequately characterized in the literature and in genomic databases so that the complete wild type sequence of the target region is known, as well as the identity and location of both clinically silent and clinically important mutations and polymorphisms.

 

Evidence of Compliance:

  • Records of literature references for wild type sequence and reported mutations and polymorphisms

 

REFERENCES

1)    Clinical and Laboratory Standards Institute (CLSI), Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline. CLSI document MM9-A (ISBN 1-56238-558-3). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004

 

MOL.34921         Sequencing Assay Optimization                

Phase II 

Sequencing assays are optimized to ensure a readable signal throughout the length of the target region and ready detection of sequence variants, especially those in heterozygous state. 

 

NOTE: Sequencing assays differ from most other molecular pathology assays in that many targets (individual nucleotides) are examined at once, rather than addressing a discrete nucleotide mutation site. Assay procedures must assure that each of these targets is visualized adequately to produce an unequivocal sequence readout, whether this is done by manual or automated methods. Heterozygous point mutations in particular may be overlooked if the signals are low or unequal. One approach to preventing this problem is to perform sequencing in both

 

  • Written procedure for performing sequencing assays

MOL.34929         Sequencing Data Criteria              

Phase I 

Criteria are established for the acceptance and interpretation of primary sequencing data. 

 

NOTE: Criteria for acceptance and interpretation of sequencing data must include correct assignments for non- polymorphic positions, definition of the sequencing region, criteria for peak intensity, baseline fluctuation, signal-to-noise ratio and peak shapes.

 

MOL.34931         Sense/Antisense Sequence         

Phase I 

The sequence of sense and antisense strands are determined for heterozygous templates, rare alleles or rare combinations of alleles. 

 

NOTE: The sequence of sense and antisense strands must be determined for rare alleles and rare combinations of alleles. The sequence of sense and antisense strands should be determined for heterozygous templates. If only one strand is sequenced for heterozygous templates, validation must show that sequencing of only one strand will consistently yield accurate sequence assignments. If assignments are routinely based on only one strand, periodic confirmation of complementary strands is recommended.

 

Evidence of Compliance:

  • Written procedure detailing how the sequence of sense and antisense strands are determined for heterozygous templates, rare alleles or rare combinations of alleles AND
  • Records showing sequencing of sense and antisense strand OR records of validation for sequence assignments based on one strand

 

MOL.34932         Sequence Interpretation Guidelines       

Phase I 

The laboratory follows professional guidelines for interpretation of sequence variation. 

  

NOTE: The laboratory should have an algorithm for decision-making in interpretation of pathogenic variants, benign variants and variants of unknown clinical significance.

 

REFERENCES

1)    ACMG Standards and Guidelines for Clinical Laboratories, http://www.acmg.net

 

Next Generation Sequencing 

  

Next Generation Sequencing (NGS) incorporates two processes: (1) the analytical wet bench process of sample and library preparation and sequence generation and (2) the bioinformatics process or pipeline of sequence alignment, annotation and variant calling. These two processes are inextricably linked as the output from each process supports the optimization of the other. The large volumes of data produced by NGS platforms put substantial demands on laboratories in terms of the requirements for documentation, validation, quality control and assurance, monitoring, data storage, as well as assessment and implementation of new technology and software releases. 

  

Inspector Instructions:

  • Sampling of next generation sequencing policies and procedures

 

Analytical Wet Bench Process 

 

MOL.34934         Wet Bench Process Documentation         

Phase I 

The laboratory documents the analytical wet bench process used to generate next generation sequencing data, including sample and library preparation as well as sequence generation. 

 

  • NOTE: Documentation to support the clinical operation must include:
  • Methods for converting DNA or RNA into a next-generation sequencing library, if applicable
  • Methods and enrichment reagents for targeted sequencing of multi-gene panels and exomes, if applicable
  • Methods for indexing (barcoding) and sample pooling, if applicable
  • Sequencing platform and manufacturing (or vendor) versions of sequencing reagents flow cells or chips
  • Analytical run parameters including base quality per read position, read lengths and read configuration (example, pair end or mate pair)
  • “Expected” analytical performance characteristics per vendor specifications and actual laboratory experience (example, minimal thresholds for base quality per read position and numbers of reads passing filters)
  • Metrics and QC parameters for optimal performance

 

Evidence of Compliance:

  • Policy or procedure that describes the laboratory’s method for such documentation AND
  • Documentation that complies with the policy or procedure
  • Repeatability and precision for the types of variants assayed (e.g. single nucleotide variants, insertions and deletions, homopolymer or repetitive sequences).
  • Interference by clinically relevant pseudogenes and other sequences highly homologous to the target must be determined and documented.
  • Sequencing error rates (i.e. false positives and false negatives) for variants assayed must be determined and documented using an alternative method which may include an alternate
  • NGS chemistry.
  • Indexing (barcoding) and sample pooling methods must be validated to ensure that individual sample identity is maintained throughout the analytical wet bench process.

 

Evidence of Compliance:

Records of validation and revalidation studies

 

MOL.34938         Quality Assurance Program          

Phase I 

The laboratory has a documented Quality Assurance (QA) program for the analytical wet bench process used to support the analysis, interpretation and reporting of next generation sequencing based results. 

 

NOTE: Procedures are in place for corrective measures should one or more components in the analytical wet bench process fail laboratory-determined quality assurance standards.

 

Evidence of Compliance:

Records of Quality Assurance program results and corrective measures, as required

 

MOL.34940         Confirmatory Testing      

Phase I 

The laboratory has a policy for when confirmatory testing of identified or reported variants will be determined by an alternative method. 

 

NOTE: The laboratory maintains an ongoing record of the sensitivity, specificity, false positives, false negatives, reproducibility and repeatability of results and compares these with data obtained during the validation process.

 

Evidence of Compliance:

  • Policy or procedure that describes the indications for confirmatory testing

 

MOL.34944         Patient Reports                 

Phase I 

The specific methods, instrument(s) and reagents are traceable for each patient report. 

 

MOL.34948         Monitoring of Upgrades                

Phase I 

The laboratory has a policy for monitoring and implementing upgrades to instruments, sequencing chemistries and reagents or kits used to generate next generation sequence data. 

 

Evidence of Compliance:

  • Documentation that demonstrates compliance with the policy or procedure

 

MOL.34952         Sequence Variants – Interpretation/Reporting  

Phase I 

Interpretation and reporting of sequence variants takes into consideration professional organizations’ recommendations and guidelines. 

 

NOTE: The laboratory should have a decision making process for classifying and interpreting pathogenic variants, potential pathogenic variants, benign variants, and variants of unknown clinical significance.

 

Evidence of Compliance:

  • Policy or procedure that describes the process used for assessment of sequence variations for potential pathogenicity

 

MOL.34954         Clinically Significant Genetic Findings    

Phase I 

The laboratory has a policy regarding reporting of clinically significant genetic findings unrelated to the clinical purpose for testing. 

 

NOTE: Gene panel(s), exome, transcriptome, and genome sequencing may yield unexpected (or incidental) clinically significant genetic findings unrelated to the disorder for which the patient is undergoing testing.

 

MOL.34956         Alternative Performance Assessment    

Phase I 

Alternative Performance Assessment for Next Generation Sequencing is part of the All 

Common Checklist: See COM.01500 Alternative Performance Assessment. 

 

NOTE: For sequencing based testing (including Sanger, pyrosequencing or next generation) alternative assessment may be performed by method and specimen type.

 

Bioinformatics Pipeline for NGS 

07.31.2012

NOTE: A bioinformatics process or pipeline includes all algorithms, software, scripts, database packages, reference sequences, and databases whether in-house developed, vendor-developed and/or supported or open source. Flow diagrams may be helpful in providing a graphical overview of processes. 

 

  • Documentation used to support clinical operations must include:
  • The individual applications used, with versions and appropriate command line flags or other configuration items that deviate from the standard, baseline installation
  • Additional scripts or steps used to connect discrete applications
  • Description of input and output data files or information in each process step
  • Metrics and QC parameters for optimal performance
  • Criteria for variant calling

 

Evidence of Compliance:

  • Policy or procedure that describes the laboratory’s method for such documentation

 

MOL.34960         Bioinformatics Process – Validation/Revalidation           Phase I 

The laboratory has validated the bioinformatics process or pipeline(s) and revalidates after any changes or upgrades are made to any components used to generate next generation sequencing data. 

 

NOTE: Validation of the bioinformatics process or pipeline(s) must describe required performance characteristics of the entire bioinformatics process or pipeline(s) and individual process steps. Revalidation may cover all or a subset of steps in the process or pipeline(s), depending on the extent of the modification. Acceptance criteria for the bioinformatics process or pipeline(s) must be established.

 

  • The validation report should include test scripts, test datasets, and other elements used in the validation.
  • Validation must describe the analytical target sequence (examples: exons, genes, exomes, genomes, and transcriptomes). The ability of the bioinformatics process or pipeline(s) to assemble or align the target sequence and generate a variant data set must be described.
  • Validations must determine and document analytical sensitivity, specificity, reproducibility and precision for the types of variants assayed (e.g. single nucleotide variants, insertions and deletions, homopolymer or repetitive sequences).
  • Interference by clinically relevant pseudogenes and other sequences highly homologous to the target must be determined and documented.
  • Bioinformatics process or pipeline(s) error rates (i.e. false positive and false negatives) for variants assayed must be determined and documented
  • Indexing (bar coding) and sample pooling methods must be validated to ensure that individual sample identity is maintained throughout the bioinformatics process or pipeline(s).

Evidence of Compliance:

  • Records of validation and revalidation studies

NOTE: Procedures are in place for corrective measures should one or more components in the bioinformatics process or pipeline fail laboratory-determined quality assurance standards.

 

Evidence of Compliance:

  • Records of Quality Assurance program results and corrective measures

 

MOL.34964         Bioinformatics Process/Pipeline – Updates         

Phase I 

The laboratory has a policy for monitoring and implementing patch-releases, upgrades, and other updates to the bioinformatics process or pipeline. 

 

Evidence of Compliance:

  • Procedure defining process for monitoring patch-releases, upgrades and updates AND
  • Documentation of monitoring activities AND
  • Dates of implementation, as appropriate

 

MOL.34966         Input Storage     

Phase I 

The laboratory has a policy regarding the storage of input, intermediate and final data files generated in the bioinformatics analyses performed on patient samples. 

 

NOTE: This policy must include: which input, intermediate and final data files are retained and which may be purged after a period of time after a final report has been generated. Selection of files for retention should support primary results generated and re-analysis at a later date if requested or required. This policy must be in accordance with local, state or national requirements for storage of data.

 

MOL.34968         Version Traceability        

Phase I 

The specific version(s) of the bioinformatics process or pipeline(s) used to generate next generation sequencing data are traceable for each patient report. 

 

NOTE: Details of the version of the bioinformatics process or pipeline used to generate each patient report may utilize a laboratory-specific system that refers to the entire process or pipeline, as well as changes to the versions of the individual components within the process or pipeline.

 

MOL.34970         Exception Log     

Phase I 

The laboratory maintains an exception log for patient cases where steps used in the bioinformatics process or pipeline deviate from laboratory SOPs. 

 

NOTE: The log file must include documentation of each deviation and reason(s) for undertaking, with sign-off by the laboratory director or designee(s). The exception log should retain links to the patient case.

 

Evidence of Compliance:

  • Documentation of review of the exception log by the laboratory director AND
  • Documentation of any issues and corrective actions taken as a result of these reviews

 

MOL.34972         Data Transfer Confidentiality Policy        

Phase I 

There are procedures in place to ensure that internal and external storage and transfer of sequencing data provides reasonable confidentiality and security, and conforms to patient confidentiality requirements. 

 

NOTE: It is recognized that laboratories may transfer data to external laboratories and other service providers for storage and analysis. This may include data storage and analysis through cloud-based computing.

 

Procedures to ensure confidentiality might include message security, system and user authentication, activity logs, encryption, and access restrictions.

 

Sequencing

Inspector Instructions:

 

  • Sampling of sequencing policies and procedures
  • How does your laboratory ensure individual nucleotides are visualized adequately?
  • How does your laboratory interpret sequence variation?

 

MOL.34914         Gene Information            

Phase II 

There is adequate information about the gene being tested regarding its wild type sequence and reported mutations and polymorphisms. 

 

NOTE: DNA sequencing assays should be reserved for those disease genes that have been adequately characterized in the literature and in genomic databases so that the complete wild type sequence of the target region is known, as well as the identity and location of both clinically silent and clinically important mutations and polymorphisms.

 

Evidence of Compliance:

  • Records of literature references for wild type sequence and reported mutations and polymorphisms

 

REFERENCES

1)    Clinical and Laboratory Standards Institute (CLSI), Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved

Guideline. CLSI document MM9-A (ISBN 1-56238-558-3). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite

1400, Wayne, Pennsylvania 19087-1898 USA, 2004

 

MOL.34921         Sequencing Assay Optimization                

Phase II 

Sequencing assays are optimized to ensure a readable signal throughout the length of the target region and ready detection of sequence variants, especially those in heterozygous state. 

 

NOTE: Sequencing assays differ from most other molecular pathology assays in that many targets (individual nucleotides) are examined at once, rather than addressing a discrete nucleotide mutation site. Assay procedures must assure that each of these targets is visualized adequately to produce an unequivocal sequence readout, whether this is done by manual or automated methods. Heterozygous point mutations in particular may be overlooked if the signals are low or unequal. One approach to preventing this problem is to perform sequencing in both

 

MOL.34929         Sequencing Data Criteria              

Phase I 

Criteria are established for the acceptance and interpretation of primary sequencing data. 

 

NOTE: Criteria for acceptance and interpretation of sequencing data must include correct assignments for non polymorphic positions, definition of the sequencing region, criteria for peak intensity, baseline fluctuation, signal-to-noise ratio and peak shapes.

 

 


 

 


Richard Resnick
Posted: Thursday, August 2, 2012 9:10 AM
Joined: 3/19/2011
Posts: 2


Thanks Gerry. It's a welcomed advance we've all been waiting for!

R


 
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