Sanger Sequencing Test Report

Molecule Tutorials - Herong's Tutorial Examples

∟Sanger Sequencing Test Report

This section provides a Sanger Sequencing test report example with a chromatogram. The Sanger Sequencing test is used to validate the result of a NGS test.

Sanger sequencing is still considered as the “gold standard” for validating DNA sequences, because it has a 99.99% base accuracy.

Below are two related reports for the same patient.

1. Blood Tumor whole Transcriptome Sequencing Report As you can see, this report identifies a single mutation on a single gene TP53 from tumor tissue.

Report Date: 07/30/2021
Clinical Diagnosis: ALL
Tumor Tissue: Tissue

Summary 
=======
Type            Tier     Mutation              Clinical Significance
-------------   ------   --------              ---------------------
Gene Mutation   Tier I   TP53 D281_R282>DQTW   Strong Significance with ALL

Appendix
========
Gene  Chr:Start      Ntchange            Amino_Acids     Vaf
----  ---------      --------            -----------     ___
TP53  chr17:7577093  c.844_845insAGACCT  D281_R282>DQTM  80%

2. Mutation Site Validation Report

Report Date: 09/20/2021
Clinical Diagnosis: ALL
Germline Tissue: Saliva

Gene  Mutation             VAF[1]  Reference Site           Validation[2]
----  --------             ------  --------------           ----------
      NM_000546:exon8:
TP53  c.844_845insAGACCT:  80%     TGGGAGAGACC[]GGCGCACAGA  Negative
      p.D281_R282>DQTW

Notes:
  [1] VAF was from previous NGS test
  [2] Validation is from this PCR test

This report looks very confusing. The first 4 columns describe a TP53 mutation with a VAF of 80%. But the last column says "Negative"!

You need to read the footnotes to understand what this report is trying to tell you:

Doctor ordered a NGS on tumor tissue first on 07/30/2021, which identified a TP53 D281_R282>DQTW mutation.
Doctor then ordered a PCR (Sanger Sequencing) test on germline tissue (saliva) to validate if the TP53 mutation was inherited.

Fortunately, the TP53 mutation was found in germline tissue. So it is not inherited. It seems to be limited in cancel cells only.

The Sanger Sequencing chromatogram given at the end of the second report confirms the above understanding.

Sanger Sequencing Chromatogram Validation - Negative

As you can see, the report DNA sequence fragment matches with the reference sequence. The insertion of "AGACCT" at position 845 (identified by the red bar in the middle of the chromatogram) reported from the NGS test on tumor tissue was not found here on germline tissue.

The above reports also give us a good example on how to read mutation notations:

NM_000546:exon8:c.844_845insAGACCT:p.D281_R282>DQTW

Where: 
  c.844_845insAGACCT - Describes the DNA mutation 
  p.D281_R282>DQTW   - Described the protein mutation

Mutation site:
  TGGGAGAGACC[]GGCGCACAGA

It is very easy to understand what happened the gene:

TP53 gene sequence mutation: c.844_845insAGACCT
                          844      845
  Reference: ...TGGGAGAGAC C[      ]G GCGCACAGA...
  Sample:    ...TGGGAGAGAC C[AGACCT]G GCGCACAGA...

But it is not so easy to understand what happened the protein, because the report uses the non-standard ">" notation. It seems to me that ">" is equivalent to "delins".

TP53 protein sequence mutation: p.D281_R282>DQTW
  Amino acids "DR" at positions 281 and 282 are replaced by "DQTW",
  which is equivalent to "R282>QTW".

The best way to understand the protein mutation is to derive it from the DNA mutation as shown below:

At the DNA level: c.844_845insAGACCT
                          844      845
  Reference: ...TGGGAGAGAC C[      ]G GCGCACAGA...
  Sample:    ...TGGGAGAGAC C[AGACCT]G GCGCACAGA...

Translate it to protein: 
  Reference
  ---------
                              844       845
  DNA:       ...T GGG AGA GAC C[        ]GG CGC ACA GA...
                          --- ------------- ---
  Protein:                Asp      Arg      Arg
                           D        R        R
                          281      282      283  

  Sample
  ------
                               844      845
  DNA:       ...T GGG AGA GAC C[AG ACT T]GG CGC ACA GA
                          --- ---- --- ---- --- 
  Protein:                Asp  Gln Thr  Trp Arg
                           D    Q   T    W   R        
                          281               283

Protein mutation: 
  D281_R282 replaced by "DQTW"
  or R282 replaced by "QTW"