G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

High-throughput full-length single-cell RNA-seq automation.

Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit-based workflow. The protocols take 3-5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative ( www.humancellatlas.org ).

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.