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Probe stringency, sequencing limits and cost per result

15 min

  • Predict how stringency changes nucleic-acid probe specificity
  • Limit a negative sequencing report to the regions and variant types the assay covers
  • Calculate cost per reportable result with a consistent denominator

Read the full reference

Try first

Try first

A labeled probe gives signal from its target gene and also from a related pseudogene that differs from the target by a few bases. Which change to the hybridization conditions reduces the pseudogene signal?

The next section explains it.

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Stringency sets how closely a probe must match

A nucleic-acid probe binds a complementary sequence. Stringency describes how closely a duplex must match to stay bound under the reaction and wash conditions.1

ChangeEffect on stringencyMismatched duplexes
Higher temperatureRaisesFewer survive
Lower salt concentrationRaisesFewer survive
More formamideRaisesFewer survive
Lower temperature or higher saltLowersMore survive

Raising stringency removes signal from related sequences, such as pseudogenes, that differ from the target by a few bases.1,2

A negative sequencing report has edges

A targeted sequencing assay is validated for stated regions and stated variant types, for example single-nucleotide variants and small insertions and deletions. A negative report covers only those.3,4

  • Coverage is judged region by region. A high average depth for the whole panel can hide one exon with no usable reads.
  • Uncovered regions are named. The report states which targeted regions did not meet the assay's quality limits, and the laboratory fills them in by a validated method, such as Sanger sequencing, when its procedure calls for it.4
  • Variant types outside validation stay unassessed. A panel validated for small variants does not assess large deletions or duplications.

Cost per reportable result

Cost per reportable result = costs assigned to the test for a period ÷ reportable patient results in that period5

  • The numerator holds every cost the laboratory assigns to the test, such as reagents, controls, calibrators, repeats, labor and service.
  • The denominator holds only patient results that were reported. Control runs, calibrators and repeats that produced no further patient result stay out of it.

Controls and repeats raise the cost per reportable result, since they add cost and no results. Comparing two instruments on this figure works only when both use the same numerator and the same denominator.5

References
  1. Gibriel A. Effect of target length on specificity and sensitivity of oligonucleotide microarrays: a comparison between dendrimer and modified PCR based labelling methods. Open Biochem J. 2014;8:11-20. doi:10.2174/1874091X01408010011
  2. Rifai N, Chiu RWK, Young I, Burnham CAD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023.
  3. Clinical and Laboratory Standards Institute. Human Genetic and Genomic Testing Using Traditional and High-Throughput Nucleic Acid Sequencing Methods. 3rd ed. CLSI guideline MM09. Clinical and Laboratory Standards Institute; 2023. Accessed September 26, 2026.
  4. Rehm HL, Bale SJ, Bayrak-Toydemir P, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med. 2013;15(9):733-747. doi:10.1038/gim.2013.92
  5. Harmening DM, ed. Laboratory Management: Principles and Processes. 4th ed. D.H. Publishing & Consulting; 2020.

Watch one

A chemistry test's costs for March are:

  • reagents $6,480
  • controls and calibrators $1,020
  • assigned labor $2,700
  • service contract share $600

The analyzer logged 4,000 runs for the test that month. Of these, 360 were control runs and 140 were repeats that produced no additional patient result. What is the cost per reportable result?

  1. Add the costs: $6,480 + $1,020 + $2,700 + $600 = $10,800.

    The numerator holds every cost assigned to the test for the period.

  2. Find the reportable results: 4,000 − 360 − 140 = 3,500.

    The denominator counts only patient results that were reported.

  3. Divide: $10,800 ÷ 3,500 = $3.0857.

    The formula divides assigned cost by reportable patient results.

  4. Round: $3.09 per reportable result.

    Money is reported to the cent, rounded once at the end.

  5. Check against cost per run: $10,800 ÷ 4,000 = $2.70, lower than $3.09, as expected.

    Cost per reportable result is always higher than cost per run when controls and repeats exist.

The cost per reportable result is $3.09.

Your turn

Problem 1 of 3

A hybridization probe gives signal from a related sequence that differs from its target by a few bases. Which change to the wash conditions reduces that signal while the matched target stays bound?

Incorrect. A lower temperature lowers stringency and lets more mismatched duplexes survive, so the signal from the related sequence would rise.

Incorrect. Higher salt concentration lowers stringency, so duplexes with a few mismatches stay bound alongside the matched target.

Correct. A higher temperature raises stringency. Duplexes with a few mismatched bases come apart, and the fully matched probe-target duplex survives.

Hint
  1. The related sequence forms a duplex with a few mismatched bases.
  2. Decide which way each change moves stringency.
  3. Higher stringency pulls apart imperfect duplexes first.

Review Hybridization and nucleic-acid separation

Problem 2 of 3

A targeted sequencing panel finds no variant in the ordered gene. The mean depth across the panel is 650×. The quality report shows that exon 7 of that gene has no usable reads. What does the report say about exon 7?

With no usable reads, exon 7 has no result. The report names it as uncovered, and a validated fill-in method such as Sanger sequencing can close the gap.

The mean depth comes from every other region. Exon 7 contributed no reads, so a variant there would go undetected.

Used high average depth to exclude an uncovered variant

Average depth summarizes the whole assay and can hide a clinically important exon with no usable reads. A negative report for a region or variant type outside the validated coverage can miss a real variant.

A statement about the whole gene includes exon 7, which was never read. The note on depth makes the gap harder to see.

Hint
  1. Average depth summarizes the whole panel.
  2. Look at the depth in exon 7 itself.

Review High-throughput sequencing

Problem 3 of 3

A test's assigned costs for one quarter are $18,600. The analyzer logged 6,000 runs, of which 420 were control runs and 380 were repeats that produced no additional patient result. What is the cost per reportable result?

Dividing $18,600 by 6,000 counts the control runs and repeats as reportable results.

Counted control and repeat runs as reportable results

The denominator counts reportable patient results. Adding 60 control and repeat runs to 300 reportable results divides $1,200 by 360 and gives $3.33 per result. The cost per reportable result is $1,200 ÷ 300 = $4.00.

Dividing $18,600 by 5,580 removes the control runs and leaves the 380 repeats in the denominator. Repeats that produced no patient result stay out too.

Reportable results are 6,000 − 420 − 380 = 5,200, and $18,600 ÷ 5,200 = $3.5769, which rounds to $3.58.

Review Cost and turnaround

Use it

  • A targeted sequencing panel for an inherited colorectal cancer syndrome is run on a woman (MRN 0072915).
  • The panel is validated for single-nucleotide variants and small insertions and deletions.
  • No variant is called in the ordered genes. Mean depth is 820×.
  • Exon 3 of MLH1 has no reads above the assay's quality limits.
  • The requisition notes a relative with a large deletion in MSH2.
  • This month the panel's assigned costs were $52,800. The laboratory reported 120 patient results, and it also sequenced 8 control samples and 12 library repeats.
Decision 1 of 3

What can the report say about MLH1?

Exon 3 had no usable reads, so the gene was not fully assessed. A variant in exon 3 would go undetected, and the high mean depth does not change that.

Used high average depth to exclude an uncovered variant

Average depth summarizes the whole assay and can hide a clinically important exon with no usable reads. A negative report for a region or variant type outside the validated coverage can miss a real variant.

The report covers what was read. Naming exon 3 shows the gap, and the procedure decides whether a validated fill-in method such as Sanger sequencing closes it.

All other targeted regions met the quality limits and have results. The gap is limited to exon 3.

Review High-throughput sequencing

Decision 2 of 3

What does the report say about the family's MSH2 deletion?

The panel was validated for small variants only. The report states that large deletions were not assessed, and testing by a method validated for them follows the laboratory's referral procedure.

Coverage of MSH2 supports the small-variant calls there. Large deletions are a variant type this panel was never validated to detect.

Leaving the deletion unmentioned lets the negative report read as covering it. The limit belongs on the report.

Review High-throughput sequencing

Decision 3 of 3

What is this month's cost per reportable result for the panel?

Dividing $52,800 by 140 counts the 8 controls and 12 repeats as reportable results. They produced no patient result.

Counted control and repeat runs as reportable results

The denominator counts reportable patient results. Adding 60 control and repeat runs to 300 reportable results divides $1,200 by 360 and gives $3.33 per result. The cost per reportable result is $1,200 ÷ 300 = $4.00.

Only the 120 reported patient results go in the denominator: $52,800 ÷ 120 = $440.00. The controls and repeats are already in the $52,800.

Dividing $52,800 by 132 counts the 12 repeats as reportable results. A repeat replaces a failed library and adds no patient result.

Review Cost and turnaround

The clue that settles this case is what the assay was validated to read. Exon 3 of MLH1 has no usable reads, and large deletions are outside the panel's variant types. The report names both limits, so a negative result is read for what it covers. The controls and repeats that kept the run reliable add to the cost and add no reportable results.

Keep

Sources checked