Clustering techniques are widely used in the analysis of large data sets to group together samples with similar properties. For example, clustering is often used in the field of single-cell ...RNA-sequencing in order to identify different cell types present in a tissue sample. There are many algorithms for performing clustering and the results can vary substantially. In particular, the number of groups present in a data set is often unknown and the number of clusters identified by an algorithm can change based on the parameters used. To explore and examine the impact of varying clustering resolution we present clustering trees. This visualisation shows the relationships between clusters at multiple resolutions allowing researchers to see how samples move as the number of clusters increases. In addition, meta-information can be overlaid on the tree to inform the choice of resolution and guide in identification of clusters. We illustrate the features of clustering trees using a series of simulations as well as two real examples, the classical iris dataset and a complex single-cell RNA-sequencing dataset. Clustering trees can be produced using the clustree R package available from CRAN (https://CRAN.R-project.org/package = clustree) and developed on GitHub (https://github.com/lazappi/clustree).
As single-cell RNA sequencing (scRNA-seq) technologies have rapidly developed, so have analysis methods. Many methods have been tested, developed, and validated using simulated datasets. ...Unfortunately, current simulations are often poorly documented, their similarity to real data is not demonstrated, or reproducible code is not available. Here, we present the Splatter Bioconductor package for simple, reproducible, and well-documented simulation of scRNA-seq data. Splatter provides an interface to multiple simulation methods including Splat, our own simulation, based on a gamma-Poisson distribution. Splat can simulate single populations of cells, populations with multiple cell types, or differentiation paths.
DNA methylation is one of the most commonly studied epigenetic modifications due to its role in both disease and development. The Illumina HumanMethylation450 BeadChip is a cost-effective way to ...profile >450 000 CpGs across the human genome, making it a popular platform for profiling DNA methylation. Here we introduce missMethyl, an R package with a suite of tools for performing normalization, removal of unwanted variation in differential methylation analysis, differential variability testing and gene set analysis for the 450K array.
missMethyl is an R package available from the Bioconductor project at www.bioconductor.org.
alicia.oshlack@mcri.edu.au
Supplementary data are available at Bioinformatics online.
As single-cell RNA-sequencing (scRNA-seq) datasets have become more widespread the number of tools designed to analyse these data has dramatically increased. Navigating the vast sea of tools now ...available is becoming increasingly challenging for researchers. In order to better facilitate selection of appropriate analysis tools we have created the scRNA-tools database (www.scRNA-tools.org) to catalogue and curate analysis tools as they become available. Our database collects a range of information on each scRNA-seq analysis tool and categorises them according to the analysis tasks they perform. Exploration of this database gives insights into the areas of rapid development of analysis methods for scRNA-seq data. We see that many tools perform tasks specific to scRNA-seq analysis, particularly clustering and ordering of cells. We also find that the scRNA-seq community embraces an open-source and open-science approach, with most tools available under open-source licenses and preprints being extensively used as a means to describe methods. The scRNA-tools database provides a valuable resource for researchers embarking on scRNA-seq analysis and records the growth of the field over time.
The fine detail provided by sequencing-based transcriptome surveys suggests that RNA-seq is likely to become the platform of choice for interrogating steady state RNA. In order to discover ...biologically important changes in expression, we show that normalization continues to be an essential step in the analysis. We outline a simple and effective method for performing normalization and show dramatically improved results for inferring differential expression in simulated and publicly available data sets.
DNA methylation is one of the most commonly studied epigenetic marks, due to its role in disease and development. Illumina methylation arrays have been extensively used to measure methylation across ...the human genome. Methylation array analysis has primarily focused on preprocessing, normalization, and identification of differentially methylated CpGs and regions. GOmeth and GOregion are new methods for performing unbiased gene set testing following differential methylation analysis. Benchmarking analyses demonstrate GOmeth outperforms other approaches, and GOregion is the first method for gene set testing of differentially methylated regions. Both methods are publicly available in the missMethyl Bioconductor R package.
Background: Short tandem repeats are an important source of genetic variation. They are highly mutable and repeat expansions are associated dozens of human disorders, such as Huntington's disease and ...spinocerebellar ataxias. Technical advantages in sequencing technology have made it possible to analyse these repeats at large scale; however, accurate genotyping is still a challenging task. We compared four different short tandem repeats genotyping tools on whole exome sequencing data to determine their genotyping performance and limits, which will aid other researchers in choosing a suitable tool and parameters for analysis.
Methods: The analysis was performed on the Simons Simplex Collection dataset, where we used a novel method of evaluation with accuracy determined by the rate of homozygous calls on the X chromosome of male samples. In total we analysed 433 samples and around a million genotypes for evaluating tools on whole exome sequencing data.
Results: We determined a relatively good performance of all tools when genotyping repeats of 3-6 bp in length, which could be improved with coverage and quality score filtering. However, genotyping homopolymers was challenging for all tools and a high error rate was present across different thresholds of coverage and quality scores. Interestingly, dinucleotide repeats displayed a high error rate as well, which was found to be mainly caused by the AC/TG repeats. Overall, LobSTR was able to make the most calls and was also the fastest tool, while RepeatSeq and HipSTR exhibited the lowest heterozygous error rate at low coverage.
Conclusions: All tools have different strengths and weaknesses and the choice may depend on the application. In this analysis we demonstrated the effect of using different filtering parameters and offered recommendations based on the trade-off between the best accuracy of genotyping and the highest number of calls.
DNA methylation is the most widely studied epigenetic mark and is known to be essential to normal development and frequently disrupted in disease. The Illumina HumanMethylation450 BeadChip assays the ...methylation status of CpGs at 485,577 sites across the genome. Here we present Subset-quantile Within Array Normalization (SWAN), a new method that substantially improves the results from this platform by reducing technical variation within and between arrays. SWAN is available in the minfi Bioconductor package.
We present GOseq, an application for performing Gene Ontology (GO) analysis on RNA-seq data. GO analysis is widely used to reduce complexity and highlight biological processes in genome-wide ...expression studies, but standard methods give biased results on RNA-seq data due to over-detection of differential expression for long and highly expressed transcripts. Application of GOseq to a prostate cancer data set shows that GOseq dramatically changes the results, highlighting categories more consistent with the known biology.
The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in ...proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.
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