ena-STUDY-University Hospital Mannheim-21-07-2017-20:26:28:928-443
ccfDNA Seq of the NEUTAX study
Targeted Amplicon Sequencing of cell-free circulating DNA of 8 patients with metastatic prostate cancer enrolled in the prospective NEUTAX study was performed.The extraction of cfDNA, as well as the subsequent amplification, sequencing, and analyses were performed at the European Genome and Diagnostics Center at GATC Biotech (GATC Biotech, Constance, Germany) by investigators who worked with pseudonymous samples and did not have access to clinical data. Targeted sequencing was performed using the GATC LIQUID ONCOPANEL on DNA extracted from 4 ml plasma. The GATC LIQUID ONCOPANEL uses multiple amplicons to interrogate mutations/hotspots in 50 oncogenes, tumor suppressors and drug resistance markers. Single molecular droplet PCR was used to amplify the target genes. Subsequently the PCR products were sequenced on the Illumina MiSeq platform using paired end sequencing with a read length of 2 x 150 bp and an approximately target coverage of 10.000x. For data analysis, the base quality of each sequence read was inspected. Low quality calls were removed before proceeding with further processing. Using a sliding window approach, bases with low quality were removed from the 3' and 5' ends. Bases were removed if the average phred quality was below 25. Finally only mate pairs (forward and reverse read) were used for the next analysis step. Mapping to the reference sequence/database (human genome 19) was done using BWA (Li et al, Bioinformatics, 2009) with default parameters. Percentage of reads in categories on target were calculated based on the number of reads mapping to entire reference. Single nucleotide variants (SNV) were detected using LoFreq (Wilm et al Nucleic Acid Research, 2012). Insertions and deletions (InDel) were detected using Platypus software (Rimmer et al, Nature Genetics, 2014). We excluded identical variants identified in more than 2 patients, as these were deemed most likely as technical artifacts.
Targeted Amplicon Sequencing of cell-free circulating DNA of 8 patients with metastatic prostate cancer enrolled in the prospective NEUTAX study was performed.The extraction of cfDNA, as well as the subsequent amplification, sequencing, and analyses were performed at the European Genome and Diagnostics Center at GATC Biotech (GATC Biotech, Constance, Germany) by investigators who worked with pseudonymous samples and did not have access to clinical data. Targeted sequencing was performed using the GATC LIQUID ONCOPANEL on DNA extracted from 4 ml plasma. The GATC LIQUID ONCOPANEL uses multiple amplicons to interrogate mutations/hotspots in 50 oncogenes, tumor suppressors and drug resistance markers. Single molecular droplet PCR was used to amplify the target genes. Subsequently the PCR products were sequenced on the Illumina MiSeq platform using paired end sequencing with a read length of 2 x 150 bp and an approximately target coverage of 10.000x. For data analysis, the base quality of each sequence read was inspected. Low quality calls were removed before proceeding with further processing. Using a sliding window approach, bases with low quality were removed from the 3' and 5' ends. Bases were removed if the average phred quality was below 25. Finally only mate pairs (forward and reverse read) were used for the next analysis step. Mapping to the reference sequence/database (human genome 19) was done using BWA (Li et al, Bioinformatics, 2009) with default parameters. Percentage of reads in categories on target were calculated based on the number of reads mapping to entire reference. Single nucleotide variants (SNV) were detected using LoFreq (Wilm et al Nucleic Acid Research, 2012). Insertions and deletions (InDel) were detected using Platypus software (Rimmer et al, Nature Genetics, 2014). We excluded identical variants identified in more than 2 patients, as these were deemed most likely as technical artifacts.