Comparison of different methods for variant calling.

<p>Mutations identified in matched frozen and FFPE tissue of CRC liver metastases detected with (A, B) Genome Analysis Toolkit (GATK) Unified Genotyper (C, D) Samtools mpileup/Bcftools and (E, F) Somatic variant caller. Green color represents FFPE samples, red represents frozen, color intensities represent number of non-synonymous coding mutations per gene.</p

KRAS mutations identified by sanger sequencing compared to deep amplicon sequencing analyzed with different variant calling tools.

<p>UG, Unified Genotyper pipeline; SAM, Samtools mpileup/Bcftools pipeline, SVC, Somatic Variant Caller; NA, not available</p><p>KRAS mutations identified by sanger sequencing compared to deep amplicon sequencing analyzed with different variant calling tools.</p

Paired frozen and FFPE samples of CRC liver metastases have a high concordance of mutations in hotspot cancer genes.

<p>(A) GATK Unified Genotyper variant calling pipeline was used to identify non-synonymous coding mutations in FFPE (green) and frozen samples (red). (B) Venn-Diagram of non-synonymous coding mutations identified in FFPE and frozen samples. (C) Representative images of reads mapped to the site of BRAF V600E mutation identified in FFPE but not in frozen tissue of patient 09, displayed with the Integrative Genomics Viewer. (D) Variant frequency of selected mutations and estimated tumor cell content analyzing FFPE samples.</p

Amplicon Sequencing identifies hot-spot mutations in CRC metastases.

<p>(A) Sequencing analysis workflow. Sequence alignment files underwent local-realignment around Indels, left alignment and base quality score recalibration. After variant calling with GATK Unified Genotyper, annotation and effect prediction of detected variants was done using SnpEff. Raw variants of all samples were filtered by custom parameters with SnpSift. Variants included in the 1000 Genomes Project data were excluded to only obtain somatic mutations in cancer. (B) High frequency of TP53 and APC mutations among somatic mutations identified in CRC liver metastases (frozen and FFPE tissue). Colored fields represent presence of a nonsynonymous coding SNP (blue), a mutation leading to a stop-codon (grey) or a frameshift mutation (orange). Bars sum up mutations present in each patient (vertical bars) or each mutated gene (horizontal bars). Of note, some genes contain more than one mutation.</p

Sequencing statistics of all patient samples.

<p>Sequencing statistics of all patient samples.</p

Depth of Sequencing correlates with DNA quality.

<p>(A) Sample preparation workflow. DNA was isolated from fresh frozen or FFPE CRC liver metastasis resection specimens with Qiagen Blood and Tissue or FFPE kit, respectively. Frozen samples then directly underwent sequencing library preparation, pooling of libraries, quality control and sequencing. FFPE samples were additionally tested for DNA quality by qPCR. Library quality was tested with Bioanalyzer. For samples with low amounts of correctly sized DNA amplicons (fragments at 310bp), new libraries were prepared with higher starting DNA concentrations and re-analyzed with Bioanalyzer. Samples with yet low amounts of DNA with correct size and highly fragmented DNA were excluded. (B) ΔCq-values of quality control PCR indicate poor sample quality. DNA concentration of fragments between 250bp and 450bp after library preparation was calculated with Agilent Bioanalyzer and plotted against ΔCq values of FFPE quality control PCR. (C) higher ΔCq-values correlate with lower mean depth of sequencing. (D) Coverage distribution of amplicons from all paired FFPE and frozen samples, normalized to total sample coverage. Frozen samples had a mean depth of 4,622, FFPE samples 1,852.</p

Additional file 1 of Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Additional file 1