Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors

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Abstract

<p class="first" id="P1">Large-scale single-cell RNA sequencing (scRNA-seq) datasets that are produced in different laboratories and at different times contain batch effects that could compromise integration and interpretation of these data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known, or the same, across batches. We present a strategy for batch correction that is based on the detection of mutual nearest neighbours (MNN) in the high-dimensional expression space. Our approach does not rely on pre-defined or equal population compositions across batches, and only requires that a subset of the population be shared between batches. We demonstrate the superiority of our approach over existing methods using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect correction method scales to large numbers of cells. </p>

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featureCounts: An efficient general-purpose program for assigning sequence reads to genomic features

, , (2013)

Next-generation sequencing technologies generate millions of short sequence reads, which are usually aligned to a reference genome. In many applications, the key information required for downstream analysis is the number of reads mapping to each genomic feature, for example to each exon or each gene. The process of counting reads is called read summarization. Read summarization is required for a great variety of genomic analyses but has so far received relatively little attention in the literature. We present featureCounts, a read summarization program suitable for counting reads generated from either RNA or genomic DNA sequencing experiments. featureCounts implements highly efficient chromosome hashing and feature blocking techniques. It is considerably faster than existing methods (by an order of magnitude for gene-level summarization) and requires far less computer memory. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications. featureCounts is available under GNU General Public License as part of the Subread (http://subread.sourceforge.net) or Rsubread (http://www.bioconductor.org) software packages.

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A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure.

Maayan Baron, Adrian Veres, Samuel Wolock … (2016)

Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.

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A Single-Cell Transcriptome Atlas of the Human Pancreas

Mauro J. Muraro, Gitanjali Dharmadhikari, Dominic Grün … (2016)

Summary To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.