PMC5993729_01
a Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Thermo Fisher Scientific Inc.), and the sequencing results were analyzed using SeqScape software version 2.5 (Applied Biosystems, Thermo Fisher Scientific Inc.). The primers used for BRCA2 exon 2 amplification and sequencing were as follows: forward (F) 5'-AGGAATATCGTAGGTAAAAATG-3' and reverse (R) 5'-CTGGATTTATACACACATAAGG-3'. Because of the low allele frequency (16%), the presence of the c.18_28delAGAGAGGCCAA variant gives a sequencing signal that is indistinguishable from background noise. b The results of the patient's genome obtained by NGS of the BRCA genes are reported; the c.18_28delAGAGAGGCCAA variant is highlighted (blue). Variant calling and the sequencing depth and quality were obtained using Amplicon Suite software (SmartSeq s.r.l., Novara, Italy), as highlighted by the arrow. c An extract of the total sequences carrying the c.18_28delAGAGAGGCCAA allele is shown (the deletion is indicated by the dotted line). The different colors (blue and gray) show the direction of sequencing (paired-end sequencing)
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Fig1
Sanger sequencing, which was used to confirm the presence of the c.18_28delAGAGAGGCCAA allele, did not reveal this allele (Fig. 1).
We used capillary electrophoresis to verify the presence of the c.18_28delAGAGAGGCCAA allele.. Although it was difficult to distinguish the results because of this method's lower resolution, amplification of the somatic mutated DNA gives two PCR products (b): the 218-bp peak of the WT allele and 207-bp peak corresponding to the BRCA2 c.18_28delAGAGAGGCCAA allele compared to the size marker (a). Normalized and shifted melting curves (c) and normalized and temperature-shifted difference plots (d) of the c.18_28delAGAGAGGCCAA allele are shown. Melting profile evaluation of the patient shows a specific melting behavior, as observed in both the normalized and the temperature-shifted and difference plots compared to the FT samples (n = 10) that do not carry this variant. Each experiment is reported in duplicate. The same forward primer used for sequencing was used for PCR-HRMA, while the PCR-HRMA-reverse primer was 5'-TCATTAGGGAGATACATATGGA-3'. The PCR-HRMA primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3) and certified as high molecular-quality products via HPLC purification (Eurofin MWG Operon, Ebersberg, Germany)
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Fig2