scholarly journals ChimeraMiner: An Improved Chimeric Read Detection Pipeline and Its Application in Single Cell Sequencing

2019 ◽  
Vol 20 (8) ◽  
pp. 1953
Author(s):  
Na Lu ◽  
Junji Li ◽  
Changwei Bi ◽  
Jing Guo ◽  
Yuhan Tao ◽  
...  
Keyword(s):  
Single Cell ◽  
Dna Polymerase ◽  
Processing Time ◽  
Structural Variation ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Superior Performance ◽  
High Fidelity ◽  
Sequencing Data ◽  
Single Cell Sequencing

As the most widely-used single cell whole genome amplification (WGA) approach, multiple displacement amplification (MDA) has a superior performance, due to the high-fidelity and processivity of phi29 DNA polymerase. However, chimeric reads, generated in MDA, cause severe disruption in many single-cell studies. Herein, we constructed ChimeraMiner, an improved chimeric read detection pipeline for analyzing the sequencing data of MDA and classified the chimeric sequences. Two datasets (MDA1 and MDA2) were used for evaluating and comparing the efficiency of ChimeraMiner and previous pipeline. Under the same hardware condition, ChimeraMiner spent only 43.4% (43.8% for MDA1 and 43.0% for MDA2) processing time. Respectively, 24.4 million (6.31%) read pairs out of 773 million reads, and 17.5 million (6.62%) read pairs out of 528 million reads were accurately classified as chimeras by ChimeraMiner. In addition to finding 83.60% (17,639,371) chimeras, which were detected by previous pipelines, ChimeraMiner screened 6,736,168 novel chimeras, most of which were missed by the previous pipeline. Applying in single-cell datasets, all three types of chimera were discovered in each dataset, which introduced plenty of false positives in structural variation (SV) detection. The identification and filtration of chimeras by ChimeraMiner removed most of the false positive SVs (83.8%). ChimeraMiner revealed improved efficiency in discovering chimeric reads, and is promising to be widely used in single-cell sequencing.

scholarly journals PhyDOSE: Design of Follow-up Single-cell Sequencing Experiments of Tumors

2020 ◽  
Author(s):  
Leah Weber ◽  
Nuraini Aguse ◽  
Nicholas Chia ◽  
Mohammed El-Kebir
Keyword(s):  
Single Cell ◽  
Retrospective Analysis ◽  
High Fidelity ◽  
Sequencing Data ◽  
Single Cell Sequencing ◽  
Bulk Data ◽  
Sequencing Experiment ◽  
Tumor Phylogeny ◽  
Number Of Cells

AbstractThe combination of bulk and single-cell DNA sequencing data of the same tumor enables the inference of high-fidelity phylogenies that form the input to many important downstream analyses in cancer genomics. While many studies simultaneously perform bulk and single-cell sequencing, some studies have analyzed initial bulk data to identify which mutations to target in a follow-up single-cell sequencing experiment, thereby decreasing cost. Bulk data provide an additional untapped source of valuable information, composed of candidate phylogenies and associated clonal prevalence. Here, we introduce PhyDOSE, a method that uses this information to strategically optimize the design of follow-up single cell experiments. Underpinning our method is the observation that only a small number of clones uniquely distinguish one candidate tree from all other trees. We incorporate distinguishing features into a probabilistic model that infers the number of cells to sequence so as to confidently reconstruct the phylogeny of the tumor. We validate PhyDOSE using simulations and a retrospective analysis of a leukemia patient, concluding that PhyDOSE’s computed number of cells resolves tree ambiguity even in the presence of typical single-cell sequencing errors. We also conduct a retrospective analysis on an acute myeloid leukemia cohort, demonstrating the potential to achieve similar results with a significant reduction in the number of cells sequenced. In a prospective analysis, we demonstrate that only a small number of cells suffice to disambiguate the solution space of trees in a recent lung cancer cohort. In summary, PhyDOSE proposes cost-efficient single-cell sequencing experiments that yield high-fidelity phylogenies, which will improve downstream analyses aimed at deepening our understanding of cancer biology.Author summaryCancer development in a patient can be explained using a phylogeny — a tree that describes the evolutionary history of a tumor and has therapeutic implications. A tumor phylogeny is constructed from sequencing data, commonly obtained using either bulk or single-cell DNA sequencing technology. The accuracy of tumor phylogeny inference increases when both types of data are used, but single-cell sequencing may become prohibitively costly with increasing number of cells. Here, we propose a method that uses bulk sequencing data to guide the design of a follow-up single-cell sequencing experiment. Our results suggest that PhyDOSE provides a significant decrease in the number of cells to sequence compared to the number of cells sequenced in existing studies. The ability to make informed decisions based on prior data can help reduce the cost of follow-up single cell sequencing experiments of tumors, improving accuracy of tumor phylogeny inference and ultimately getting us closer to understanding and treating cancer.

484 Bioturing browser: interactively explore public single cell sequencing data

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A520-A520
Author(s):  
Son Pham ◽  
Tri Le ◽  
Tan Phan ◽  
Minh Pham ◽  
Huy Nguyen ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cell ◽  
Expression Profiles ◽  
Meta Analysis ◽  
Cell Types ◽  
Sequencing Data ◽  
Single Cell Sequencing ◽  
Data Formats ◽  
Cancer Types ◽  
Cell Data

BackgroundSingle-cell sequencing technology has opened an unprecedented ability to interrogate cancer. It reveals significant insights into the intratumoral heterogeneity, metastasis, therapeutic resistance, which facilitates target discovery and validation in cancer treatment. With rapid advancements in throughput and strategies, a particular immuno-oncology study can produce multi-omics profiles for several thousands of individual cells. This overflow of single-cell data poses formidable challenges, including standardizing data formats across studies, performing reanalysis for individual datasets and meta-analysis.MethodsN/AResultsWe present BioTuring Browser, an interactive platform for accessing and reanalyzing published single-cell omics data. The platform is currently hosting a curated database of more than 10 million cells from 247 projects, covering more than 120 immune cell types and subtypes, and 15 different cancer types. All data are processed and annotated with standardized labels of cell types, diseases, therapeutic responses, etc. to be instantly accessed and explored in a uniform visualization and analytics interface. Based on this massive curated database, BioTuring Browser supports searching similar expression profiles, querying a target across datasets and automatic cell type annotation. The platform supports single-cell RNA-seq, CITE-seq and TCR-seq data. BioTuring Browser is now available for download at www.bioturing.com.ConclusionsN/A

scholarly journals Effective clustering for single cell sequencing cancer data

2019 ◽  
Author(s):  
Simone Ciccolella ◽  
Murray Patterson ◽  
Paola Bonizzoni ◽  
Gianluca Della Vedova
Keyword(s):  
Single Cell ◽  
Categorical Data ◽  
Euclidean Distance ◽  
Missing Values ◽  
False Negative ◽  
Ground Truth ◽  
Sequencing Data ◽  
Large Space ◽  
Cancer Data ◽  
Single Cell Sequencing

AbstractBackgroundSingle cell sequencing (SCS) technologies provide a level of resolution that makes it indispensable for inferring from a sequenced tumor, evolutionary trees or phylogenies representing an accumulation of cancerous mutations. A drawback of SCS is elevated false negative and missing value rates, resulting in a large space of possible solutions, which in turn makes infeasible using some approaches and tools. While this has not inhibited the development of methods for inferring phylogenies from SCS data, the continuing increase in size and resolution of these data begin to put a strain on such methods.One possible solution is to reduce the size of an SCS instance — usually represented as a matrix of presence, absence and missing values of the mutations found in the different sequenced cells — and infer the tree from this reduced-size instance. Previous approaches have used k-means to this end, clustering groups of mutations and/or cells, and using these means as the reduced instance. Such an approach typically uses the Euclidean distance for computing means. However, since the values in these matrices are of a categorical nature (having the three categories: present, absent and missing), we explore techniques for clustering categorical data — commonly used in data mining and machine learning — to SCS data, with this goal in mind.ResultsIn this work, we present a new clustering procedure aimed at clustering categorical vector, or matrix data — here representing SCS instances, called celluloid. We demonstrate that celluloid clusters mutations with high precision: never pairing too many mutations that are unrelated in the ground truth, but also obtains accurate results in terms of the phylogeny inferred downstream from the reduced instance produced by this method.Finally, we demonstrate the usefulness of a clustering step by applying the entire pipeline (clustering + inference method) to a real dataset, showing a significant reduction in the runtime, raising considerably the upper bound on the size of SCS instances which can be solved in practice.AvailabilityOur approach, celluloid: clustering single cell sequencing data around centroids is available at https://github.com/AlgoLab/celluloid/ under an MIT license.

scholarly journals K-mer counting with low memory consumption enables fast clustering of single-cell sequencing data without read alignment

2019 ◽  
Author(s):  
Christina Huan Shi ◽  
Kevin Y. Yip
Keyword(s):  
Single Cell ◽  
State Of The Art ◽  
Rna Seq ◽  
Sequencing Data ◽  
Memory Consumption ◽  
Analysis Pipeline ◽  
Cell Clusters ◽  
Single Cell Sequencing ◽  
Sequencing Errors ◽  
Full Analysis

AbstractK-mer counting has many applications in sequencing data processing and analysis. However, sequencing errors can produce many false k-mers that substantially increase the memory requirement during counting. We propose a fast k-mer counting method, CQF-deNoise, which has a novel component for dynamically identifying and removing false k-mers while preserving counting accuracy. Compared with four state-of-the-art k-mer counting methods, CQF-deNoise consumed 49-76% less memory than the second best method, but still ran competitively fast. The k-mer counts from CQF-deNoise produced cell clusters from single-cell RNA-seq data highly consistent with CellRanger but required only 5% of the running time at the same memory consumption, suggesting that CQF-deNoise can be used for a preview of cell clusters for an early detection of potential data problems, before running a much more time-consuming full analysis pipeline.

scholarly journals Evaluation of single-cell classifiers for single-cell RNA sequencing data sets

2019 ◽  
Vol 21 (5) ◽  
pp. 1581-1595 ◽  
Author(s):  
Xinlei Zhao ◽  
Shuang Wu ◽  
Nan Fang ◽  
Xiao Sun ◽  
Jue Fan
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Reference Data ◽  
Predictive Accuracy ◽  
Cell Types ◽  
Superior Performance ◽  
Marker Genes ◽  
Data Sets ◽  
Sequencing Data ◽  
Single Cell Rna Sequencing

Abstract Single-cell RNA sequencing (scRNA-seq) has been rapidly developing and widely applied in biological and medical research. Identification of cell types in scRNA-seq data sets is an essential step before in-depth investigations of their functional and pathological roles. However, the conventional workflow based on clustering and marker genes is not scalable for an increasingly large number of scRNA-seq data sets due to complicated procedures and manual annotation. Therefore, a number of tools have been developed recently to predict cell types in new data sets using reference data sets. These methods have not been generally adapted due to a lack of tool benchmarking and user guidance. In this article, we performed a comprehensive and impartial evaluation of nine classification software tools specifically designed for scRNA-seq data sets. Results showed that Seurat based on random forest, SingleR based on correlation analysis and CaSTLe based on XGBoost performed better than others. A simple ensemble voting of all tools can improve the predictive accuracy. Under nonideal situations, such as small-sized and class-imbalanced reference data sets, tools based on cluster-level similarities have superior performance. However, even with the function of assigning ‘unassigned’ labels, it is still challenging to catch novel cell types by solely using any of the single-cell classifiers. This article provides a guideline for researchers to select and apply suitable classification tools in their analysis workflows and sheds some lights on potential direction of future improvement on classification tools.

scholarly journals Tumor Copy Number Deconvolution Integrating Bulk and Single-Cell Sequencing Data

2020 ◽  
Vol 27 (4) ◽  
pp. 565-598 ◽  
Author(s):  
Haoyun Lei ◽  
Bochuan Lyu ◽  
E. Michael Gertz ◽  
Alejandro A. Schäffer ◽  
Xulian Shi ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Sequencing Data ◽  
Single Cell Sequencing

scholarly journals rCASC: reproducible classification analysis of single-cell sequencing data

GigaScience ◽  
2019 ◽  
Vol 8 (9) ◽  
Author(s):  
Luca Alessandrì ◽  
Francesca Cordero ◽  
Marco Beccuti ◽  
Maddalena Arigoni ◽  
Martina Olivero ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cellular Heterogeneity ◽  
Cell Subpopulation ◽  
Integrated Analysis ◽  
Specific Gene ◽  
Sequencing Data ◽  
Single Cell Sequencing ◽  
Cell Stability ◽  
Reproducible Analysis

Abstract Background Single-cell RNA sequencing is essential for investigating cellular heterogeneity and highlighting cell subpopulation-specific signatures. Single-cell sequencing applications have spread from conventional RNA sequencing to epigenomics, e.g., ATAC-seq. Many related algorithms and tools have been developed, but few computational workflows provide analysis flexibility while also achieving functional (i.e., information about the data and the tools used are saved as metadata) and computational reproducibility (i.e., a real image of the computational environment used to generate the data is stored) through a user-friendly environment. Findings rCASC is a modular workflow providing an integrated analysis environment (from count generation to cell subpopulation identification) exploiting Docker containerization to achieve both functional and computational reproducibility in data analysis. Hence, rCASC provides preprocessing tools to remove low-quality cells and/or specific bias, e.g., cell cycle. Subpopulation discovery can instead be achieved using different clustering techniques based on different distance metrics. Cluster quality is then estimated through the new metric "cell stability score" (CSS), which describes the stability of a cell in a cluster as a consequence of a perturbation induced by removing a random set of cells from the cell population. CSS provides better cluster robustness information than the silhouette metric. Moreover, rCASC's tools can identify cluster-specific gene signatures. Conclusions rCASC is a modular workflow with new features that could help researchers define cell subpopulations and detect subpopulation-specific markers. It uses Docker for ease of installation and to achieve a computation-reproducible analysis. A Java GUI is provided to welcome users without computational skills in R.

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