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.

SPAdes—St. Petersburg genome Assembler—was originally developed for de novo assembly of genome sequencing data produced for cultivated microbial isolates and for single‐cell genomic DNA sequencing. With time, the functionality of SPAdes was extended to enable assembly of IonTorrent data, as well as hybrid assembly from short and long reads (PacBio and Oxford Nanopore). In this article we present protocols for five different assembly pipelines that comprise the SPAdes package and that are used for assembly of metagenomes and transcriptomes as well as assembly of putative plasmids and biosynthetic gene clusters from whole‐genome sequencing and metagenomic datasets. In addition, we present guidelines for understanding results with use cases for each pipeline, and several additional support protocols that help in using SPAdes properly. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assembling isolate bacterial datasets Basic Protocol 2: Assembling metagenomic datasets Basic Protocol 3: Assembling sets of putative plasmids Basic Protocol 4: Assembling transcriptomes Basic Protocol 5: Assembling putative biosynthetic gene clusters Support Protocol 1: Installing SPAdes Support Protocol 2: Providing input via command line Support Protocol 3: Providing input data via YAML format Support Protocol 4: Restarting previous run Support Protocol 5: Determining strand‐specificity of RNA‐seq data

Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing "microbial dark matter" that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license.

MotivationThe emergence of high-throughput sequencing technologies revolutionized genomics in early 2000s. The next revolution came with the era of long-read sequencing. These technological advances along with novel computational approaches became the next step towards the automatic pipelines capable to assemble nearly complete mammalian-size genomes.ResultsIn this manuscript, we demonstrate performance of the state-of-the-art genome assembly software on six eukaryotic datasets sequenced using different technologies. To evaluate the results, we developed QUAST-LG—a tool that compares large genomic de novo assemblies against reference sequences and computes relevant quality metrics. Since genomes generally cannot be reconstructed completely due to complex repeat patterns and low coverage regions, we introduce a concept of upper bound assembly for a given genome and set of reads, and compute theoretical limits on assembly correctness and completeness. Using QUAST-LG, we show how close the assemblies are to the theoretical optimum, and how far this optimum is from the finished reference.Availability and implementation http://cab.spbu.ru/software/quast-lg Supplementary information Supplementary data are available at Bioinformatics online.

supplementary data are available at Bioinformatics online.