Additional file 6: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)
Creators: name: Kjällquist, Una
name: Erlandsson, Rikard
name: Tobin, Nicholas
name: Alkodsi, Amjad
name: Ullah, Ikram
name: Stålhammar, Gustav
name: Karlsson, Eva
name: Hatschek, Thomas
name: Hartman, Johan
name: Linnarsson, Sten
name: Bergh, Jonas
Publisher: name: figshare
Year: 2018
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Evolutionary Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Table S3. Control experiment. To investigate the rate of false positive mutations as well as the loss of alleles introduced by the amplification process we designed a control experiment as follows: Genomic DNA from a healthy individual was extracted from whole blood using the PAXgene Blood DNA kit (Qiagen). The DNA was of good quality (&gt; 58 kB fragment length) and of high concentration (&gt; 300 ng/uL). This DNA was diluted 1:100 and 1:1000 and 6 ng respectively 0.6 ng was subjected to WGA, exome enriched, sequenced and analyzed as described in (Fig. 2a) using an unamplified sample as ‘germline’ control. In the control exome experiment with 6 ng input gDNA, we found no (zero) variant positions, indicating that the false discovery rate was negligible when the amount of starting material was in the range of 6 ng or more. In the control exome experiment with 0.6 ng input gDNA, we found 27 amino acid altering mutations that passed our SNV calling criteria. This indicates that false positive SNVs can start to appear as the amount of input material is reduced. We used this higher rate of false positives to estimate the false discovery rate (FDR) in samples with input less than 6 ng. Note that no tumor sample was amplified from less than 1.2 ng DNA. Allelic drop out or loss of heterozygosity due to biased amplification towards one of the alleles will be challenging to distinguish from true LOH. As expected, in the control exomes no LOH was detected. The fraction of called LOH positions was 0.6% and 1% of all variant positions in the 6 ng and 0.6 ng control experiments respectively, and these positions did not form continuous regions. In most tumor samples, we could clearly distinguish regions of true LOH, as blocks of SNPs with LOH calls. Outside these regions, any observed LOH could be assumed to be artefactual. We therefore determined the fraction of LOH calls in regions without signs of true LOH in the tumor samples, as a measure of false negative calls (allelic dropout). In the primary tumor samples we found 1.36% (0.1%- 8.3%) false negatives, whereas in the metastatic samples this fraction was 4.88% (0%–16.25%). However, three of the samples (metastases of patients P1, P4 and P10) were of poor quality showing a very noisy pattern of variant allele frequencies, most likely due to DNA degradation and low concentration. In these three samples the false negative discovery rate could not be calculated but could be approximated by assuming that there were no true LOH calls at all, resulting in a conservative estimate of 43–87% false negative calls in these three samples. We could not identify LOH independently in these samples, but we used the SNP phase test described in (Additional file 4: Figure S1a-b) to confirm or reject the existence of LOH alterations in the metastatic lesion concordant with the primary tumor. Although the median false negative SNV rate was less than 1%, the metastatic samples of three patients (P1, P4 and P10) suffered a rate exceeding 40%, presumably due to allelic dropout during amplification. The median false discovery rate was 1%, and did not exceed 6% for any patient. (PDF 398 kb)