Benchmarking of de novo assembly algorithms for Nanopore data reveals optimal performance of OLC approaches.

Improved DNA sequencing methods have transformed the field of genomics over the last decade. This has become possible due to the development of inexpensive short read sequencing technologies which have now resulted in three generations of sequencing platforms. More recently, a new fourth generation of Nanopore based single molecule sequencing technology, was developed based on MinION(®) sequencer which is portable, inexpensive and fast. It is capable of generating reads of length greater than 100 kb. Though it has many specific advantages, the two major limitations of the MinION reads are high error rates and the need for the development of downstream pipelines. The algorithms for error correction have already emerged, while development of pipelines is still at nascent stage. In this study, we benchmarked available assembler algorithms to find an appropriate framework that can efficiently assemble Nanopore sequenced reads. To address this, we employed genome-scale Nanopore sequenced datasets available for E. coli and yeast genomes respectively. In order to comprehensively evaluate multiple algorithmic frameworks, we included assemblers based on de Bruijn graphs (Velvet and ABySS), Overlap Layout Consensus (OLC) (Celera) and Greedy extension (SSAKE) approaches. We analyzed the quality, accuracy of the assemblies as well as the computational performance of each of the assemblers included in our benchmark. Our analysis unveiled that OLC-based algorithm, Celera, could generate a high quality assembly with ten times higher N50 & mean contig values as well as one-fifth the number of total number of contigs compared to other tools. Celera was also found to exhibit an average genome coverage of 12 % in E. coli dataset and 70 % in Yeast dataset as well as relatively lesser run times. In contrast, de Bruijn graph based assemblers Velvet and ABySS generated the assemblies of moderate quality, in less time when there is no limitation on the memory allocation, while greedy extension based algorithm SSAKE generated an assembly of very poor quality but with genome coverage of 90 % on yeast dataset. OLC can be considered as a favorable algorithmic framework for the development of assembler tools for Nanopore-based data, followed by de Bruijn based algorithms as they consume relatively less or similar run times as OLC-based algorithms for generating assembly, irrespective of the memory allocated for the task. However, few improvements must be made to the existing de Bruijn implementations in order to generate an assembly with reasonable quality. Our findings should help in stimulating the development of novel assemblers for handling Nanopore sequence data.

Cherukuri Y ，Janga SC 《BMC GENOMICS》

Assembly of long error-prone reads using de Bruijn graphs.

The recent breakthroughs in assembling long error-prone reads were based on the overlap-layout-consensus (OLC) approach and did not utilize the strengths of the alternative de Bruijn graph approach to genome assembly. Moreover, these studies often assume that applications of the de Bruijn graph approach are limited to short and accurate reads and that the OLC approach is the only practical paradigm for assembling long error-prone reads. We show how to generalize de Bruijn graphs for assembling long error-prone reads and describe the ABruijn assembler, which combines the de Bruijn graph and the OLC approaches and results in accurate genome reconstructions.

Lin Y ，Yuan J ，Kolmogorov M ，Shen MW ，Chaisson M ，Pevzner PA ... -  《-》

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer.

Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology.

Istace B ，Friedrich A ，d'Agata L ，Faye S ，Payen E ，Beluche O ，Caradec C ，Davidas S ，Cruaud C ，Liti G ，Lemainque A ，Engelen S ，Wincker P ，Schacherer J ，Aury JM ... -  《GigaScience》

Clover: a clustering-oriented de novo assembler for Illumina sequences.

Next-generation sequencing technologies revolutionized genomics by producing high-throughput reads at low cost, and this progress has prompted the recent development of de novo assemblers. Multiple assembly methods based on de Bruijn graph have been shown to be efficient for Illumina reads. However, the sequencing errors generated by the sequencer complicate analysis of de novo assembly and influence the quality of downstream genomic researches. In this paper, we develop a de Bruijn assembler, called Clover (clustering-oriented de novo assembler), that utilizes a novel k-mer clustering approach from the overlap-layout-consensus concept to deal with the sequencing errors generated by the Illumina platform. We further evaluate Clover's performance against several de Bruijn graph assemblers (ABySS, SOAPdenovo, SPAdes and Velvet), overlap-layout-consensus assemblers (Bambus2, CABOG and MSR-CA) and string graph assembler (SGA) on three datasets (Staphylococcus aureus, Rhodobacter sphaeroides and human chromosome 14). The results show that Clover achieves a superior assembly quality in terms of corrected N50 and E-size while remaining a significantly competitive in run time except SOAPdenovo. In addition, Clover was involved in the sequencing projects of bacterial genomes Acinetobacter baumannii TYTH-1 and Morganella morganii KT. The marvel clustering-based approach of Clover that integrates the flexibility of the overlap-layout-consensus approach and the efficiency of the de Bruijn graph method has high potential on de novo assembly. Now, Clover is freely available as open source software from https://oz.nthu.edu.tw/~d9562563/src.html .

Hsieh MF ，Lu CL ，Tang CY 《BMC BIOINFORMATICS》

FastEtch: A Fast Sketch-Based Assembler for Genomes.

De novo genome assembly describes the process of reconstructing an unknown genome from a large collection of short (or long) reads sequenced from the genome. A single run of a Next-Generation Sequencing (NGS) technology can produce billions of short reads, making genome assembly computationally demanding (both in terms of memory and time). One of the major computational steps in modern day short read assemblers involves the construction and use of a string data structure called the de Bruijn graph. In fact, a majority of short read assemblers build the complete de Bruijn graph for the set of input reads, and subsequently traverse and prune low-quality edges, in order to generate genomic "contigs"-the output of assembly. These steps of graph construction and traversal, contribute to well over 90 percent of the runtime and memory. In this paper, we present a fast algorithm, FastEtch, that uses sketching to build an approximate version of the de Bruijn graph for the purpose of generating an assembly. The algorithm uses Count-Min sketch, which is a probabilistic data structure for streaming data sets. The result is an approximate de Bruijn graph that stores information pertaining only to a selected subset of nodes that are most likely to contribute to the contig generation step. In addition, edges are not stored; instead that fraction which contribute to our contig generation are detected on-the-fly. This approximate approach is intended to significantly improve performance (both execution time and memory footprint) whilst possibly compromising on the output assembly quality. We present two main versions of the assembler-one that generates an assembly, where each contig represents a contiguous genomic region from one strand of the DNA, and another that generates an assembly, where the contigs can straddle either of the two strands of the DNA. For further scalability, we have implemented a multi-threaded parallel code. Experimental results using our algorithm conducted on E. coli, Yeast, C. elegans, and Human (Chr2 and Chr2+3) genomes show that our method yields one of the best time-memory-quality trade-offs, when compared against many state-of-the-art genome assemblers.

Ghosh P ，Kalyanaraman A 《-》