A custom amplicon-based sequencing panel covering 29 genes (Supplemental Table 2 ) known to be recurrently
mutated in cutaneous and uveal melanoma was designed and prepared applying the GeneRead
Library Prep Kit from QIAGEN® according to the manufacturer's
instructions. For adapter ligation and barcoding of individual samples, the NEBNext Ultra
DNA Library Prep Mastermix Set and NEBNext Multiplex Oligos for Illumina from New England
Biolabs were applied. Twelve samples were sequenced in parallel on an Illumina MiSeq next
generation sequencer.
Sequencing analysis was performed applying the CLC Cancer Research Workbench
from QIAGEN®. In brief, the following steps were applied. The workflow
in CLC included adapter trimming and read pair merging before mapping to the human
reference genome (hg19). Insertions and deletions as well as single nucleotide variant
detection, local realignment and primer trimming followed. Additional information was then
obtained regarding potential mutation type, known single nucleotide polymorphisms and
conservation scores by cross-referencing varying databases (COSMIC, ClinVar, dbSNP, 1000
Genomes Project, HAPMAP and PhastCons-Conservation_scores_hg19). After the CLC processing,
resulting csv files were analyzed manually. Mutations affecting the protein coding portion
of the gene were considered if predicted to result in non-synonymous amino acid changes.
To eliminate questionable low frequency background mutations calls, not uncommon in our
experience with FFPE amplicon sequencing approaches [24 (link)], mutations were reported if the overall coverage of
the mutation site was ≥30 reads, ≥15 reads reported the mutated variant
and the frequency of mutated reads was ≥15 %.
Sanger-sequencing was performed for EIF1AX exon 1 applying the
primers F-CCTCCAGCACCTACTTGGTC and R-CTGGGTGACCTGCAATCTAC as previously described
[21 (link)].
mutated in cutaneous and uveal melanoma was designed and prepared applying the GeneRead
Library Prep Kit from QIAGEN® according to the manufacturer's
instructions. For adapter ligation and barcoding of individual samples, the NEBNext Ultra
DNA Library Prep Mastermix Set and NEBNext Multiplex Oligos for Illumina from New England
Biolabs were applied. Twelve samples were sequenced in parallel on an Illumina MiSeq next
generation sequencer.
Sequencing analysis was performed applying the CLC Cancer Research Workbench
from QIAGEN®. In brief, the following steps were applied. The workflow
in CLC included adapter trimming and read pair merging before mapping to the human
reference genome (hg19). Insertions and deletions as well as single nucleotide variant
detection, local realignment and primer trimming followed. Additional information was then
obtained regarding potential mutation type, known single nucleotide polymorphisms and
conservation scores by cross-referencing varying databases (COSMIC, ClinVar, dbSNP, 1000
Genomes Project, HAPMAP and PhastCons-Conservation_scores_hg19). After the CLC processing,
resulting csv files were analyzed manually. Mutations affecting the protein coding portion
of the gene were considered if predicted to result in non-synonymous amino acid changes.
To eliminate questionable low frequency background mutations calls, not uncommon in our
experience with FFPE amplicon sequencing approaches [24 (link)], mutations were reported if the overall coverage of
the mutation site was ≥30 reads, ≥15 reads reported the mutated variant
and the frequency of mutated reads was ≥15 %.
Sanger-sequencing was performed for EIF1AX exon 1 applying the
primers F-CCTCCAGCACCTACTTGGTC and R-CTGGGTGACCTGCAATCTAC as previously described
[21 (link)].
