Normalization is a crucial step in the analysis of single-cell RNA-sequencing (scRNA-seq) counts data. Its principal objectives are reduction of systematic biases primarily introduced through technical sources and transformation of counts to make them more amenable for the application of established statistical frameworks. In the standard workflows, normalization is followed by feature selection to identify highly variable genes (HVGs) that capture most of the biologically meaningful variation across the cells. Here, we make the case for a revised workflow by proposing a simple feature selection method and showing that we can perform feature selection before normalization by relying on observed counts. We highlight that the feature selection step can be used to not only select HVGs but to also identify stable genes. We further propose a novel variance stabilization transformation inclusive residuals-based normalization method that in fact relies on the stable genes to inform the reduction of systematic biases. We demonstrate significant improvements in downstream clustering analyses through the application of our proposed methods on biological truth-known as well as simulated counts datasets. We have implemented this novel workflow for analyzing high-throughput scRNA-seq data in an R package called Piccolo.

SUMMARY: Clinical sequencing aims to identify somatic mutations in cancer cells for accurate diagnosis and treatment. However, most widely used clinical assays lack patient-matched control DNA and additional analysis is needed to distinguish somatic and unfiltered germline variants. Such computational analyses require accurate assessment of tumor cell content in individual specimens. Histological estimates often do not corroborate with results from computational methods that are primarily designed for normal-tumor matched data and can be confounded by genomic heterogeneity and presence of sub-clonal mutations. Allele-frequency-based imputation of tumor (All-FIT) is an iterative weighted least square method to estimate specimen tumor purity based on the allele frequencies of variants detected in high-depth, targeted, clinical sequencing data. Using simulated and clinical data, we demonstrate All-FIT's accuracy and improved performance against leading computational approaches, highlighting the importance of interpreting purity estimates based on expected biology of tumors. AVAILABILITY AND IMPLEMENTATION: Freely available at http://software.khiabanian-lab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Rapid progress in high-throughput sequencing (HTS) and the development of novel library preparation methods have improved the sensitivity of detecting mutations in heterogeneous samples, specifically in high-depth (> 500×) clinical applications. However, HTS methods are bounded by their technical and theoretical limitations and sequencing errors cannot be completely eliminated. Comprehensive quantification of the background noise can highlight both the efficiency and the limitations of any HTS methodology, and help differentiate true mutations at low abundance from artifacts. RESULTS: , thereby establishing error rates for 96 possible substitutions as well as four single-base and 16 double-base indels. We applied MERIT to ultra-deep sequencing data (1,300,000 ×) obtained from the amplification of multiple clinically relevant loci, and showed a significant relationship between error rates and genomic contexts. In addition to observing significant difference between transversion and transition rates, we identified variations of more than 100-fold within each error type at high sequencing depths. For instance, T >G transversions in trinucleotide GTCs occurred 133.5 ± 65.9 more often than those in ATAs. Similarly, C >T transitions in GCGs were observed at 73.8 ± 10.5 higher rate than those in TCTs. We also devised an in silico approach to determine the optimal sequencing depth, where errors occur at rates similar to those of expected true mutations. Our analyses showed that increasing sequencing depth might improve sensitivity for detecting some mutations based on their genomic context. For example, T >G rate of error in GTCs did not change when sequenced beyond 10,000 ×; in contrast, T >G rate in TTAs consistently improved even at above 500,000 ×. CONCLUSIONS: Our results demonstrate significant variation in nucleotide misincorporation rates, and suggest that genomic context should be considered for comprehensive profiling of specimen-specific and sequencing artifacts in high-depth assays. This data provide strong evidence against assigning a single allele frequency threshold to call mutations, for it can result in substantial false positive as well as false negative variants, with important clinical consequences.

BACKGROUND: Patterns of disease incidence can identify new risk factors for the disease or provide insight into the etiology. For example, allergies and infectious diseases have been shown to follow periodic temporal patterns due to seasonal changes in environmental or infectious agents. Previous work searching for seasonal or other temporal patterns in disease diagnosis rates has been limited both in the scope of the diseases examined and in the ability to distinguish unexpected seasonal patterns. Electronic Health Records (EHR) compile extensive longitudinal clinical information, constituting a unique source for discovery of trends in occurrence of disease. However, the data suffer from inherent biases that preclude an identification of temporal trends. METHODS: Motivated by observation of the biases in this data source, we developed a method (Lomb-Scargle periodograms in detrended data, LSP-detrend) to find periodic patterns by adjusting the temporal information for broad trends in incidence, as well as seasonal changes in total hospitalizations. LSP-detrend can sensitively uncover periodic temporal patterns in the corrected data and identify the significance of the trend. We apply LSP-detrend to a compilation of records from 1.5 million patients encoded by ICD-9-CM (International Classification of Diseases, Ninth Revision, Clinical Modification), including 2,805 disorders with more than 500 occurrences across a 12 year period, recorded from 1.5 million patients. RESULTS AND CONCLUSIONS: Although EHR data, and ICD-9 coded records in particular, were not created with the intention of aggregated use for research, these data can in fact be mined for periodic patterns in incidence of disease, if confounders are properly removed. Of all diagnoses, around 10% are identified as seasonal by LSP-detrend, including many known phenomena. We robustly reproduce previous findings, even for relatively rare diseases. For instance, Kawasaki disease, a rare childhood disease that has been associated with weather patterns, is detected as strongly linked with winter months. Among the novel results, we find a bi-annual increase in exacerbations of myasthenia gravis, a potentially life threatening complication of an autoimmune disease. We dissect the causes of this seasonal incidence and propose that factors predisposing patients to this event vary through the year.