DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly

Abstract

Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with~0.13 genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.

microRNA Biomarker Discovery and High-Throughput DNA Sequencing Are Possible Using Long-term Archived Serum Samples

“…The evenness in sequence distribution ( Supplementary Fig. S7) also suggests that these results could be used to identify CNVs using methods developed for low coverage sequencing (48). A limitation of the study is that the sample size in the sequencing experiments is not sufficient to identify the variation in sequencing yield or the "failure rate", if any, should we sequence all samples in the SerumBank.…”

Tumor Cell–Free DNA Copy Number Instability Predicts Therapeutic Response to Immunotherapy

“…Reads with a mapping quality score not reaching the maximum value in BWA were censored form further analysis. After correction for GC content and mapability using proprietary algorithms for cfDNA sequencing, the read counts were transformed into log2 ratios (17) and converted into Z-values based on Gaussian transformations versus a normal control group (n ¼ 126). These secondary data were then subjected to a noise-reducing proprietary bioinformatics pipeline using stochastic and statistic algorithms to calculate a final Z-score for each bin value to be within the dispersion of the normal control group (null hypothesis: equality).…”

Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium

“…One aliquot of this library was subjected to shallow wholegenome sequencing (WGS) for genome-wide copy number analysis, 34 and another aliquot was subjected to hybrid capture target enrichment (Roche NimbleGen, Madison, WI, USA) for mutation analysis. Eight libraries were equimolarly pooled per capture.…”

“…250ng DNA from each patient sample was sheared on a Covaris S2 (Covaris Inc., Woburn, MA, USA), with settings adjusted to DNA from FFPE tissue. 34 NGS libraries were prepared using KAPA Library Preparation kits (KAPA Biosystems, Inc., Wilmington, MA, USA). In short, uniquely 8-bp indexed adapters (Roche NimbleGen, Madison, WI, USA.)…”