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Aim: Whole-genome methylation sequencing carries both DNA methylation and structural variant information (single nucleotide variant [SNV]; copy number variant [CNV]); however, limited data is available on the reliability of obtaining this information simultaneously from low-input DNA using various library preparation and sequencing protocols. Methods: A HapMap NA12878 sample was sequenced with three protocols (EM-sequencing, QIA-sequencing and Swift-sequencing) and their performance was compared on CpG methylation measurement and SNV and CNV detection. Results: At low DNA input (10-25 ng), EM-sequencing was superior in almost all metrics except CNV detection where all protocols were similar. EM-sequencing captured the highest number of CpGs and true SNVs. Conclusion: EM-sequencing is suitable to detect methylation, SNVs and CNVs from single sequencing with low-input DNA.

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS., Competing Interests: The authors have declared that no competing interests exist.

Background: Quantification of circulating organ-specific cell-free DNA (cfDNA) provides a sensitive measure of ongoing cell death that could benefit evaluation of the cholestatic liver diseases primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), which lack reliable non-invasive biomarkers. Our goal in this pilot study was to determine whether liver-specific cfDNA levels are increased in PBC and PSC patients relative to controls and in advanced versus early disease, to evaluate their potential as novel disease biomarkers.Methods: Peripheral blood derived bisulfite-treated DNA was PCR amplified from patients with PBC (n = 48), PSC (n = 48) and controls (n = 96) to evaluate methylation status at 16 CpG sites reported to be specifically unmethylated in liver tissue near the genes IGF2R, ITIH4 and VTN. Amplicons were used to prepare paired end libraries which were sequenced on a MiSeq sequencer. Trimmed reads were aligned and used to determine unmethylation ratios and to calculate concentration of liver-specific cfDNA. Comparisons between groups were performed using the two-tailed Mann-Whitney Test and relationships between variables were evaluated using Pearson's Correlation.Results: Levels of liver-specific cfDNA, as measured at the 3 genetic loci, were increased in PBC and PSC patients relative to controls and in late-stage relative to early-stage patients. As well, cfDNA levels were correlated with levels of alkaline phosphatase, a commonly used biochemical test to evaluate disease severity in liver disease, in patients, but not in controls.Conclusions: cfDNA offers promise as a non-invasive liquid-biopsy to evaluate liver-specific cell-death in patients with cholestatic liver diseases. [ABSTRACT FROM AUTHOR]

Objective: To analyze methylation profiles of known preeclampsia/eclampsia (PE) candidate genes in normal (NL) and preeclamptic (PE) women at delivery.Methods: A matched case-control study comparing methylation in 79 CpG sites/33 genes from an independent gene set in maternal leukocyte DNA in PE and NL (n = 14 each) on an Illumina BeadChip platform. Replication performed on second cohort (PE = 12; NL = 32).Results: PE demonstrates differential methylation in POMC, AGT, CALCA, and DDAH1 compared with NL.Conclusion: Differential methylation in four genes associated with PE may represent a potential biomarker or an epigenetic pathophysiologic mechanism altering gene transcription. [ABSTRACT FROM AUTHOR]

Background Novel and targetable mutations are needed for improved understanding and treatment of lung cancer in never-smokers. Methods Twenty-seven lung adenocarcinomas from never-smokers were sequenced by both exome and mRNA-seq with respective normal tissues. Somatic mutations were detected and compared with pathway deregulation, tumor phenotypes and clinical outcomes. Results Although somatic mutations in DNA or mRNA ranged from hundreds to thousands in each tumor, the overlap mutations between the two were only a few to a couple of hundreds. The number of somatic mutations from either DNA or mRNA was not significantly associated with clinical variables; however, the number of overlap mutations was associated with cancer subtype. These overlap mutants were preferentially expressed in mRNA with consistently higher allele frequency in mRNA than in DNA. Ten genes (EGFR, TP53, KRAS, RPS6KB2, ATXN2, DHX9, PTPN13, SP1, SPTAN1 and MYOF) had recurrent mutations and these mutations were highly correlated with pathway deregulation and patient survival. Conclusions The recurrent mutations present in both DNA and RNA are likely the driver for tumor biology, pathway deregulation and clinical outcomes. The information may be used for patient stratification and therapeutic target development. [ABSTRACT FROM AUTHOR]

Objective: To compare genome-wide methylation profiles in maternal leukocyte DNA between normotensive and preeclamptic pregnant women at delivery. Methods: Age, body mass index matched case-control comparison of methylation at 27,578 cytosine- guanine sites in 14,495 genes in maternal leukocyte DNA in women with preeclampsia (PE; n = 14) and normotensive controls ( n = 14). Results: PE was associated with widespread differential methylation favoring hypermethylation. Pathway analysis identified the best matched process as a neuropeptide signaling pathway ( p < 10−5); best matched disease as eclampsia ( p < 9.97 × 10−20). Significantly differentially methylated genes ( GRIN2b. GABRA1. PCDHB7, and BEX1) are associated with seizures. Conclusion: Altered maternal leukocyte DNA methylation is associated with PE at delivery, and differential methylation of certain neuronal genes may explain the risk for eclampsia. [ABSTRACT FROM AUTHOR]