Journal: BMC Cancer

Article Title: Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

doi: 10.1186/1471-2407-14-13

Figure Lengend Snippet: Representative results for BRAF exon 15 mutation analysis. Sanger sequencing (A - C) , high resolution melting (HRM) analysis (D - F) , pyrosequencing (Pyro) (G - I) and immunohistochemistry (IHC) (J-L) are compared: The first column shows exemplarily p.V600E mutations, the second p.V600K mutations and the third column p.V600R mutations. In HRM, normalized and temperature shifted difference plots showing wildtype control in blue and mutant control in red. HRM can distinguish between p.V600E (red) and p.V600K (green) and p.V600R (light blue). Pyrosequencing was performed in the reverse direction with the sequence to analyze 5’- YAY TGT AGC TAG ACS AAA AYC ACC -3’. All three mutations can be detected. Immunohistochemistry shows a strong staining for p.V600E (J) but is negative for p.V600K (K) in representative melanoma sample. Pigmentation has to be clearly distinguished from a positive p.V600E staining (K) . Cross reactivity was observed for p.V600R mutation (L) in a colorectal carcinoma sample. A: adenine, C: cytosine, G: guanine, T: thymine, V: valine, E: glutamic acid, K: lysine, R: arginine.

Article Snippet: In the study of Shen and Qin (2012) a p.V600K mutation was overlooked by visual inspection but was detected using pyrosequencing data analysis software [ ].

Techniques: Mutagenesis, Sequencing, Immunohistochemistry, Control, Staining

Journal: BMC Cancer

Article Title: Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

doi: 10.1186/1471-2407-14-13

Figure Lengend Snippet: Melanoma sample showing different results of the BRAF p.V600E mutational analysis (case 30). Sanger sequencing as well as high resolution melting analysis show wildtype results ( A and B , respectively). Pyrosequencing analysis resulted in a p.V600E mutation with a 5% mutant allele frequency having low relative fluorescent units of almost 30 (C) . This result is confirmed by immunohistochemistry (D) . Next generation sequencing resulted in a 2% mutant allele frequency with a coverage rate of 181 (E) .

Article Snippet: In the study of Shen and Qin (2012) a p.V600K mutation was overlooked by visual inspection but was detected using pyrosequencing data analysis software [ ].

Techniques: Sequencing, Mutagenesis, Immunohistochemistry, Next-Generation Sequencing

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: Primer design

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Software, Sequencing, Amplification

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: Primer design. As one can see in the Design Template, there is a padding region of 300 base pairs (bp) before and after the exon where the forward and reverse primers can be designed. The amplicon includes the target region which in turn consists of the region of interest (exon and a padding of 15bp important in the splicing mechanism) and a region of 5-35 bp that is technical requirement of the 454 or Sanger respectively to give accurate sequencing results. Each primer (P1 and 2) contains a specific and a universal region (black and red regions). The specific region attaches to the gene of interest. This primer region is used during the amplification process (PCR) and becomes part of the amplicon. The universal primer used during the sequencing process hybridizes to the universal region allowing the sequencing of any amplicon.

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Amplification, Sequencing

Journal:

Article Title: Basic Principles and Technologies for Deciphering the Genetic Map of Cancer

doi: 10.1007/s00268-008-9851-y

Figure Lengend Snippet: K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software. The different intensity of the superposed bases in the Sanger chromatograms of the different patients illustrates the difficulty for an automatic call with SNP detector. On the other hand, in the 454 pyrosequencing, the mutations are successfully called by AVA. For this reason, the 454 pyrosequencing is a great discovery technology. However, the % given is not representative of the % of patients with the mutations because of factors such as heterogenous sample, heteroploidy and chromosomal loss. Genotyping with another technology then needs to be performed in each sample as well as in the matched normal tissue to determine which mutation is somatic and define the percentage of patients with each mutation.

Article Snippet: Open in a separate window Fig. 7 K-RAS mutations in codon 12 and 13 illustrated in Sanger chromatogram and 454 pyrosequencing Amplicon Variant Analysis software.

Techniques: Amplification, Variant Assay, Software, Mutagenesis