Tissue sample adequacy |
Criteria for inadequate specimen |
Testing inadequate specimens may lead to a waste of time and money or depletion of available samples. |
Check sample adequacy rigorously before testing. |
Minimum tumor content |
Request further sampling in case of inadequate samples. |
Appropriate sample handling including fixation and transportation |
Inadequate amount or tumor content can lead to false-negative test results. |
Nucleic acid extraction |
DNA quantity and quality in terms of amplifiable DNA |
DNA with suboptimal quality may inhibit sequencing reaction. |
Failed samples should be reported as such and further material might be requested with specified requirement. |
Small amount or fragmentation of DNA may lead to poor quality sequencing data with insufficient or uneven coverage and/or high duplication rate. |
Trying another validated extraction method may often helps. |
Sample identification |
Sample identity tracking throughout all steps |
Misidentification of samples could lead to incorrect patient management. |
If there is any concern about sample identity, starting over from DNA extraction may be necessary. |
Introduction of polymorphic SNP markers into gene panel and running another genotyping method with the same marker set might be helpful. |
Library preparation |
Minimum library concentration |
Poor sequencing library may lead to insufficient or uneven coverage. |
Consider modification of library preparation method or an alternative method to verify any uncertain results. |
Libraries with poor complexity or bias may result in false-negatives. False-positives may also occur due to potential amplification bias. |
Sequencing |
Criteria for minimum sequencing depth and other quality metrics (% reads mapped to target regions, % of targets with specified coverage, duplication rate) |
Inadequate coverage is associated with higher levels of uncertainty of the test results. |
Repeat sequencing with existing library or start over from DNA extraction step. |
Genomic regions with insufficient local coverage may lead to inaccurate results for variants located in those regions. |
Verification of uncertain results with another method may be helpful, especially, in case of actionable variants. |
Variant detection and review |
Variant allele frequency, local sequencing depth and quality score |
Failure to filter out sequencing artifacts may lead to false-positive results. |
Manually review of clinically important variants even if computational algorithms called no mutation on them. |
Presence of the same variant in forward and reverse strands |
Clinically important variants may sometimes be missed. |
Any ambiguous or unexpected results should be reviewed by laboratory scientists and pathologists. |
Mapping quality of sequencing reads |
Verify variants with another method, if applicable. |
Potential sequencing artifacts |
Bioinformatics |
Correct pipeline and version |
Using outdated or inadequate software can lead to false-positive or false-negative results. |
Update software on a regular basis. |
Appropriate version and build of human reference sequence |
Cross-contamination? |
Reporting |
Endorsed by an authorized competent pathologist? |
Variants with clinical significance may be reported erroneously, leading to inappropriate treatment. |
Responsible pathologists should be given enough time and opportunities for education and training. |