A new method to accurately identify single nucleotide variants using small FFPE breast samples.

Fortunato, A.; Mallo Adan, D.; Rupp, S. M.; King, L.; Hardman, T.; Lo, J.; Hall, A.; Marks, J. R.; Hwang, E. S.; Maley, C. C.

doi:10.1101/2020.10.22.350983

Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.10.22.350983

A new method to accurately identify single nucleotide variants using small FFPE breast samples.

Fortunato, A.·Mallo Adan, D.·Rupp, S. M.·King, L.·Hardman, T.·Lo, J.·Hall, A.·Marks, J. R.·Hwang, E. S.·Maley, C. C.

View original source ↗Export metadata ↓

Abstract

Most tissue collections of neoplasms are composed of formalin-fixed and paraffin-embedded (FFPE) excised tumor samples used for routine diagnostics. DNA sequencing is becoming increasingly important in cancer research and clinical management; however, it is difficult to accurately sequence DNA from FFPE samples. We developed and validated a new bioinformatic algorithm to robustly identify somatic single nucleotide variants (SNVs) from whole exome sequencing using small amounts of DNA extracted from archival FFPE samples of breast cancers. We optimized this strategy using 28 pairs of technical replicates. After optimization, the mean similarity between replicates increased 5-fold, reaching 88% (range 0-100%), with a mean of 21.4 SNVs (range 1-68) per sample, representing a markedly superior performance to existing algorithms. We found that the SNV-identification accuracy declined when there was less than 40ng of DNA available and that insertion-deletion variant calls are less reliable than single base substitutions. As the first application of the new algorithm, we compared samples of ductal carcinoma in situ (DCIS) of the breast to their adjacent invasive ductal carcinoma (IDC) samples. We observed an increased number of mutations (paired-samples sign test, p<0.05), and a higher genetic divergence in the invasive samples (paired-samples sign test, p<0.01). Our algorithm provides a significant improvement in detecting SNVs in FFPE samples over previous approaches. Key PointsO_LIThe sequencing of reduced quantities of DNA extracted from FFPE samples leads to substantial sequencing errors that require correction in order to obtain accurate detection of somatic mutations. C_LIO_LIWe developed and validated a new bioinformatic algorithm to robustly identify somatic single nucleotide variants using small amounts of DNA extracted from archival FFPE samples of breast cancers. C_LIO_LIVariant calling software packages need to be optimized to reduce the impact of sequencing errors. Our bioinformatics pipeline represents a significant methodological advance compared to the currently available bioinformatic tools used for the analysis of small FFPE samples. C_LI

Source connections

Published article ↗

Explore related subjects

genomics Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Fortunato, A., Mallo Adan, D., Rupp, S. M., King, L., Hardman, T., Lo, J., Hall, A., Marks, J. R., Hwang, E. S., Maley, C. C.. 2020-10-23. A new method to accurately identify single nucleotide variants using small FFPE breast samples.. https://doi.org/10.1101/2020.10.22.350983

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

SPACE-Tag Enables High-Throughput Spatial Chromatin Profiling With CUT&Tag

Spatial epigenomics maps chromatin states within intact tissues, yet current methods remain limited by accessibility and scale. We developed SPACE-Tag, a spatial CUT&Tag framework that converts epigenetic marks-associated DNA into polyadenylated RNA through T7 based linear amplification, enabling direct integration with standard spatial transcriptomic workflows. Applied to the mouse brain, SPACETag resolved activating and repressive chromatin landscapes, recovered region-specific gene regulatory programs across anatomical domains, and showed strong correlation with existing bulk and single cell reference data. We generated an integrated multimodal atlas of the aging mouse brain, profiling five histone modifications and the transcriptome in parallel across the adult lifespan to establish the largest spatial multimodal dataset to date. This analysis uncovered sharp regional divergence with aging in the brain: fiber tracts showed widespread transcriptional downregulation, often without nearby chromatin state transitions, whereas the isocortex showed coordinated chromatin transcriptome changes, including an H3K4me1 peak module that increased with age, was enriched at super-enhancers and linked to upregulation of neuronal plasticity regulators. These findings highlight the power of multimodal and high throughput spatial genomics in uncovering the divergent regulatory logic that governs tissue-specific vulnerability and resilience in the aging brain.

genomics↗

bioRxiv2026

Long-read DNA sequencing reveals global hypomethylation and focal hypermethylation in SDHx-deficient pheochromocytoma and paraganglioma

Pheochromocytomas and paragangliomas (PPGL) are highly heritable neuroendocrine tumors of the autonomic ganglia. Genomic DNA in most cancer cells is hypomethylated. However, DNA in PPGLs secondary to mutations in succinate dehydrogenase complex (SDHx) genes is reportedly hypermethylated due to succinate-mediated inhibition of DNA demethylases. We used nanopore long-read sequencing to characterize the methylome of tumor and white blood cell DNA from SDHx and non-SDHx PPGL patients, alongside normal adrenal medulla. We find that both SDHx and non-SDHx PPGLs are globally hypomethylated, particularly at centromeres. In normal adrenal medulla, CpG islands marked by H3K4 methylation are hypomethylated, whereas CpG islands lacking H3K4 methylation are hypermethylated. In SDHx-related PPGL, some H3K4me-marked CpG islands are hypermethylated, but most are not. We propose that DNA methylation in SDHx-related PPGL reflects the global hypomethylation generally observed in tumors, partly offset by succinate-mediated inhibition of TET DNA demethylases. Opposing effects of succinate may alter the balance between DNA methylation (indirect inhibition of the DNMT3A/B DNA methylases via inhibition of H3K4me demethylases) and DNA demethylation (inhibition of TET DNA demethylases) at active CpG islands.

genomics↗

bioRxiv2026

High piRNA abundance is a hallmark of evolutionarily persistent piRNA-producing loci.

PIWI-interacting RNAs (piRNAs) silence transposons in the germline, and the loci that produce them diverge rapidly between species. Yet, some loci retain both their genomic location and their piRNA production across large evolutionary distances, and the basis for this conservation is unclear. To identify the features that distinguish these persistent loci, we compared piRNA production at orthologous loci in three closely related mouse species, contrasting loci that produce piRNAs in all three species with those that do so in only one. Transposon content and UTR features, often linked to piRNA production, did not distinguish the two groups. Transcription extending beyond gene ends (readthrough) was more frequent at conserved loci, but did not explain conservation once the amount of piRNAs produced was taken into account. Instead, we found that the conserved loci are those that produce the most piRNAs. The same association holds true in human and two other primate species. We speculate that most piRNAs expressed in testis are tolerated molecular noise, and that only the highly expressed minority are likely to be functional.

genomics↗