The third-generation long reads sequencing technologies, such as PacBio and Nanopore, have great advantages over second-generation Illumina sequencing in de novo assembly studies. However, due to the inherent low base accuracy, third-generation sequencing data cannot be used for k-mer counting and estimating genomic profile based on k-mer frequencies. Thus, in current genome projects, second-generation data is also necessary for accurately determining genome size and other genomic characteristics. We show that corrected third-generation data can be used to count k-mer frequencies and estimate genome size reliably, in replacement of using second-generation data. Therefore, future genome projects can depend on only one sequencing technology to finish both assembly and k-mer analysis, which will largely decrease sequencing cost in both time and money. Moreover, we present a fast light-weight tool kmerfreq and use it to perform all the k-mer counting tasks in this work. We have demonstrated that corrected third-generation sequencing data can be used to estimate genome size and developed a new open-source C/C++ k-mer counting tool, kmerfreq, which is freely available at https://github.com/fanagislab/kmerfreq.
Programs based on hash tables and Burrows-Wheeler are very fast for mapping short reads to genomes but have low accuracy in the presence of mismatches and gaps. Such reads can be aligned accurately with the Smith-Waterman algorithm but it can take hours and days to map millions of reads even for bacteria genomes. We introduce a GPU program called MaxSSmap with the aim of achieving comparable accuracy to Smith-Waterman but with faster runtimes. Similar to most programs MaxSSmap identifies a local region of the genome followed by exact alignment. Instead of using hash tables or Burrows-Wheeler i…
In the last decade a number of algorithms and associated software have been developed to align next generation sequencing (NGS) reads with relevant reference genomes. The accuracy of these programs may vary significantly, especially when the NGS reads are quite different from the available reference genome. We propose a benchmark to assess accuracy of short reads mapping based on the pre-computed global alignment of related genome sequences. In this paper we propose a benchmark to assess accuracy of the short reads mapping based on the pre-computed global alignment of closely related genome se…
Long-read sequencing has enabled the de novo assembly of several mammalian genomes, but with high cost in computing. Here, we demonstrated de novo assembly of mammalian genome using long reads in an efficient and inexpensive workstation.
Genomics has become an essential technology for surveilling emerging infectious disease outbreaks. A wide range of technologies and strategies for pathogen genome enrichment and sequencing are being used by laboratories worldwide, together with different, and sometimes ad hoc, analytical procedures for generating genome sequences. As a result, public repositories now contain non-standard entries of varying quality. A standardized analytical process for consensus genome sequence determination, particularly for outbreaks such as the ongoing COVID-19 pandemic, is critical to provide a solid genom…
Background: With the fast development of next generation sequencing technologies, increasing numbers of genomes are being de novo sequenced and assembled. However, most are in fragmental and incomplete draft status, and thus it is often difficult to know the accurate genome size and repeat content. Furthermore, many genomes are highly repetitive or heterozygous, posing problems to current assemblers utilizing short reads. Therefore, it is necessary to develop efficient assembly-independent methods for accurate estimation of these genomic characteristics. Results: Here we present a framework fo…
(An updated version of this manuscript has been accepted to Scientific Reports in 2016, please refer to http://www.nature.com/articles/srep31900) The highly anticipated transition from next generation sequencing (NGS) to third generation sequencing (3GS) has been difficult primarily due to high error rates and excessive sequencing cost. The high error rates make the assembly of long erroneous reads of large genomes challenging because existing software solutions are often overwhelmed by error correction tasks. Here we report a hybrid assembly approach that simultaneously utilizes NGS and 3GS d…
While most current high-throughput DNA sequencing technologies generate short reads with low error rates, emerging sequencing technologies generate long reads with high error rates. A basic question of interest is the tradeoff between read length and error rate in terms of the information needed for the perfect assembly of the genome. Using an adversarial erasure error model, we make progress on this problem by establishing a critical read length, as a function of the genome and the error rate, above which perfect assembly is guaranteed. For several real genomes, including those from the GAGE …
Fraunhofer USA CMA（弗劳恩霍夫美国中心）；University of Maryland（马里兰大学）
In this paper we apply the structure of genomes as second-order Markov processes specified by the distributions of successive triplets of bases to two bioinformatics problems: identification of outliers in genome databases and read classification in metagenomics, using real coronavirus and adenovirus data.
Motivation: New long read sequencers promise to transform sequencing and genome assembly by producing reads tens of kilobases long. However their high error rate significantly complicates assembly and requires expensive correction steps to layout the reads using standard assembly engines. Results: We present an original and efficient spectral algorithm to layout the uncorrected nanopore reads, and its seamless integration into a straightforward overlap/layout/consensus (OLC) assembly scheme. The method is shown to assemble Oxford Nanopore reads from several bacterial genomes into good quality …
Supregraph: Enabling Information-Optimal Assembly Graph Representation of a Read Set
Department of Computer Science and Engineering, Pennsylvania State University（宾夕法尼亚州立大学计算机科学与工程系）；Huck Institutes of the Life Sciences, Pennsylvania State University（宾夕法尼亚州立大学哈克生命科学学院）
The $r$-index is a tool for compressed indexing of genomic databases for exact pattern matching, which can be used to completely align reads that perfectly match some part of a genome in the database or to find seeds for reads that do not. This paper shows how to download and install the programs ri-buildfasta and ri-align; how to call ri-buildfasta on a FASTA file to build an $r$-index for that file; and how to query that index with ri-align. Availability: The source code for these programs is released under GPLv3 and available at https://github.com/alshai/r-index .
Variant calling, the problem of estimating whether a position in a DNA sequence differs from a reference sequence, given noisy, redundant, overlapping short sequences that cover that position, is fundamental to genomics. We propose a deep averaging network designed specifically for variant calling. Our model takes into account the independence of each short input read sequence by transforming individual reads through a series of convolutional layers, limiting the communication between individual reads to averaging and concatenating operations. Training and testing on the precisionFDA Truth Cha…
We present Meraculous2, an update to the Meraculous short-read assembler that includes (1) handling of allelic variation using "bubble" structures within the de Bruijn graph, (2) improved gap closing, and (3) an improved scaffolding algorithm that produces more complete assemblies without compromising scaffolding accuracy. The speed and bandwidth efficiency of the new parallel implementation have also been substantially improved, allowing the assembly of a human genome to be accomplished in 24 hours on the JGI/NERSC Genepool system. To highlight the features of Meraculous2 we present here the …
Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium ${\it Escherichia coli}$, for $\approx$ 65$\%$ of the promoters we remain completely ignorant of their regulation. Until we have cracked this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method (Reg-Seq) linking a massively-parallel reporter assay and mass spectrometry to produce a base pair resolution dissection of more than 100 promoters in ${\it E. coli}$ in 12 different growth conditions. Firs…
Genome analysis fundamentally starts with a process known as read mapping, where sequenced fragments of an organism's genome are compared against a reference genome. Read mapping is currently a major bottleneck in the entire genome analysis pipeline, because state-of-the-art genome sequencing technologies are able to sequence a genome much faster than the computational techniques employed to analyze the genome. We describe the ongoing journey in significantly improving the performance of read mapping. We explain state-of-the-art algorithmic methods and hardware-based acceleration approaches. A…
De novo genome assembly, i.e., rebuilding the sequence of an unknown genome from redundant and erroneous short sequences, is a key but computationally intensive step in many genomics pipelines. The exponential growth of genomic data is increasing the computational demand and requires scalable, high-performance approaches. In this work, we present a novel distributed-memory algorithm that, from a string graph representation of the genome and using sparse matrices, generates the contig set, i.e., overlapping sequences that form a map representing a region of a chromosome. Using matrix abstractio…
Next-generation sequencing technology enables routine detection of bacterial pathogens for clinical diagnostics and genetic research. Whole genome sequencing has been of importance in the epidemiologic analysis of bacterial pathogens. However, few whole genome sequencing-based genotyping pipelines are available for practical applications. Here, we present the whole genome sequencing-based single nucleotide polymorphism (SNP) genotyping method and apply to the evolutionary analysis of methicillin-resistant Staphylococcus aureus. The SNP genotyping method calls genome variants using next-generat…
A crucial problem in genome assembly is the discovery and correction of misassembly errors in draft genomes. We develop a method that will enhance the quality of draft genomes by identifying and removing misassembly errors using paired short read sequence data and optical mapping data. We apply our method to various assemblies of the loblolly pine and Francisella tularensis genomes. Our results demonstrate that we detect more than 54% of extensively misassembled contigs and more than 60% of locally misassembed contigs in an assembly of Francisella tularensis, and between 31% and 100% of extens…
Genome sequencing is the basis for many modern biological and medicinal studies. With recent technological advances, metagenomics has become a problem of interest. This problem entails the analysis and reconstruction of multiple DNA sequences from different sources. Shotgun genome sequencing works by breaking up long DNA sequences into shorter segments called reads. Given this collection of reads, one would like to reconstruct the original collection of DNA sequences. For experimental design in metagenomics, it is important to understand how the minimal read length necessary for reliable recon…
High read depth can be used to assemble short sequence repeats. The existing genome assemblers fail in repetitive regions of longer than average read. I propose a new algorithm for a DNA assembly which uses the relative frequency of reads to properly reconstruct repetitive sequences. The mathematical model shows the upper limits of accuracy of the results as a function of read coverage. For high coverage, the estimation error depends linearly on repetitive sequence length and inversely proportional to the sequencing coverage. The algorithm requires high read depth, provided by the next-generat…
The advent of next-generation sequencing (NGS) technologies enables researchers to sequence complex microbial communities directly from environment. Since assembly typically produces only genome fragments, also known as contigs, instead of entire genome, it is crucial to group them into operational taxonomic units (OTUs) for further taxonomic profiling and down-streaming functional analysis. OTU clustering is also referred to as binning. We present COCACOLA, a general framework automatically bin contigs into OTUs based upon sequence composition and coverage across multiple samples. The effecti…
A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data
动机：由于细菌基因组组装的复杂性，许多生物学家仍然无法获得它。很少有软件解决方案能够使来自Illumina数据的从头基因组组装过程中的所有步骤自动化。结果如下：A5-miseq可以在膝上型计算机上从低至20倍的序列数据覆盖产生高质量的微生物基因组组装，而无需任何参数调整。A5-miseq通过自动化适配器修剪、质量过滤、错误校正、重叠群和支架生成以及错误组装检测的过程来实现这一点。与原始A5流水线不同，A5-miseq可以使用来自Illumina MiSeq的长读段，在重叠群生成期间使用读段配对信息，并包括对读段修剪的几项改进。这些改变一起导致实质上改进的组装，其比先前的方法恢复更完整的参考基因组。可用性：A5-miseq是在GPL开源许可下许可的。Mac OS X 10.6+和Linux 2.6.15+的源代码和预编译二进制文件可从http://sourceforge.net/projects/ngopt获得
The rapidly changing landscape of sequencing technologies brings new opportunities to genomics research. Longer sequence reads and higher sequence throughput coupled with ever-improving base accuracy and decreasing per-base cost is now making long reads suitable for analyzing polymorphic regions of the human genome, such as those of the human leucocyte antigen (HLA) gene complex. Here I present a simple protocol for predicting HLA signatures from whole genome shotgun (WGS) long sequencing reads, by directly streaming sequence alignments into HLAminer. The method is as simple as running minimap…
Motivation: Next generation methods of DNA sequencing produce relatively high rate of reading errors, which interfere with de novo genome assembly of newly sequenced organisms and particularly affect the quality of SNP detection important for diagnostics of many hereditary diseases. There exists a number of programs developed for correcting errors in NGS reads. Such programs utilize various approaches and are optimized for different specific tasks, but all of them are far from being able to correct all errors, especially in sequencing reads that crossing by repeats and DNA from di/polyploid eu…
Purdue University（普渡大学）；Indian Institute of Technology Bombay（印度理工学院孟买分校）；Adobe Research（Adobe 研究院）
Sequencing technologies are prone to errors, making error correction (EC) necessary for downstream applications. EC tools need to be manually configured for optimal performance. We find that the optimal parameters (e.g., k-mer size) are both tool- and dataset-dependent. Moreover, evaluating the performance (i.e., Alignment-rate or Gain) of a given tool usually relies on a reference genome, but quality reference genomes are not always available. We introduce Lerna for the automated configuration of k-mer-based EC tools. Lerna first creates a language model (LM) of the uncorrected genomic reads;…
Genome sequence analysis has enabled significant advancements in medical and scientific areas such as personalized medicine, outbreak tracing, and the understanding of evolution. Unfortunately, it is currently bottlenecked by the computational power and memory bandwidth limitations of existing systems, as many of the steps in genome sequence analysis must process a large amount of data. A major contributor to this bottleneck is approximate string matching (ASM). We propose GenASM, the first ASM acceleration framework for genome sequence analysis. We modify the underlying ASM algorithm (Bitap) …
ETH Zurich（苏黎世联邦理工学院）；Carnegie Mellon University（卡内基梅隆大学）；Bilkent University（比尔肯特大学）
Long reads produced by third-generation sequencing technologies are used to construct an assembly (i.e., the subject's genome), which is further used in downstream genome analysis. Unfortunately, long reads have high sequencing error rates and a large proportion of bps in these long reads are incorrectly identified. These errors propagate to the assembly and affect the accuracy of genome analysis. Assembly polishing algorithms minimize such error propagation by polishing or fixing errors in the assembly by using information from alignments between reads and the assembly (i.e., read-to-assembly…
