The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E + V -SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online (http://bioinf.spbau.ru/spades). It is distributed as open source software.

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A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

We report full-length draft De novo genome assemblies for 16 widely used inbred mouse strains and reveal extensive strain-specific haplotype variation. We identify and characterise 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 and 62 new coding loci were added to the reference genome annotation. Notably these genomes revealed a large previously unannotated gene ( Efcab3-like ) encoding 5,874 amino acids. Efcab3-like mutant mice display anomalies in multiple brain regions suggesting a role in the regulation of brain development.

Summary: Bacterial genomes are simpler than mammalian ones, and yet assembling the former from the data currently generated by high-throughput short-read sequencing machines still results in hundreds of contigs. To improve assembly quality, recent studies have utilized longer Pacific Biosciences (PacBio) reads or jumping libraries to connect contigs into larger scaffolds or help assemblers resolve ambiguities in repetitive regions of the genome. However, their popularity in contemporary genomic research is still limited by high cost and error rates. In this work, we explore the possibility of improving assemblies by using complete genomes from closely related species/strains. We present Ragout, a genome rearrangement approach, to address this problem. In contrast with most reference-guided algorithms, where only one reference genome is used, Ragout uses multiple references along with the evolutionary relationship among these references in order to determine the correct order of the contigs. Additionally, Ragout uses the assembly graph and multi-scale synteny blocks to reduce assembly gaps caused by small contigs from the input assembly. In simulations as well as real datasets, we believe that for common bacterial species, where many complete genome sequences from related strains have been available, the current high-throughput short-read sequencing paradigm is sufficient to obtain a single high-quality scaffold for each chromosome.Availability: The Ragout software is freely available at: https://github.com/fenderglass/Ragout.Contact: spham@salk.edu