Local de novo assembly of RAD paired-end contigs using short sequencing reads

Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.     

De novo assembly of the Pseudomonas syringae pv. syringae B728a genome using Illumina/Solexa short sequence reads

Illumina's Genome Analyzer generates ultra-short sequence reads, typically 36 nucleotides in length, and is primarily intended for resequencing. We tested the potential of this technology for de novo sequence assembly on the 6 Mbp genome of Pseudomonas syringae pv. syringae B728a with several freely available assembly software packages. Using an unpaired data set, velvet assembled >96% of the genome into contigs with an N50 length of 8289 nucleotides and an error rate of 0.33%. edena generated smaller contigs (N50 was 4192 nucleotides) and comparable error rates. ssake and vcake yielded shorter contigs with very high error rates. Assembly of paired-end sequence data carrying 400 bp inserts produced longer contigs (N50 up to 15 628 nucleotides), but with increased error rates (0.5%). Contig length and error rate were very sensitive to the choice of parameter values. Noncoding RNA genes were poorly resolved in de novo assemblies, while >90% of the protein-coding genes were assembled with 100% accuracy over their full length. This study demonstrates that, in practice, de novo assembly of 36-nucleotide reads can generate reasonably accurate assemblies from about 40 × deep sequence data sets. These draft assemblies are useful for exploring an organism's proteomic potential, at a very economic low cost.     

Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing

Background

Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood.

Results

We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results.

Conclusions

In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1859-8) contains supplementary material, which is available to authorized users.     

PERGA: A Paired-End Read Guided De Novo Assembler for Extending Contigs Using SVM and Look Ahead Approach

Since the read lengths of high throughput sequencing (HTS) technologies are short, de novo assembly which plays significant roles in many applications remains a great challenge. Most of the state-of-the-art approaches base on de Bruijn graph strategy and overlap-layout strategy. However, these approaches which depend on k-mers or read overlaps do not fully utilize information of paired-end and single-end reads when resolving branches. Since they treat all single-end reads with overlapped length larger than a fix threshold equally, they fail to use the more confident long overlapped reads for assembling and mix up with the relative short overlapped reads. Moreover, these approaches have not been special designed for handling tandem repeats (repeats occur adjacently in the genome) and they usually break down the contigs near the tandem repeats. We present PERGA (Paired-End Reads Guided Assembler), a novel sequence-reads-guided de novo assembly approach, which adopts greedy-like prediction strategy for assembling reads to contigs and scaffolds using paired-end reads and different read overlap size ranging from O _max to O _min to resolve the gaps and branches. By constructing a decision model using machine learning approach based on branch features, PERGA can determine the correct extension in 99.7% of cases. When the correct extension cannot be determined, PERGA will try to extend the contig by all feasible extensions and determine the correct extension by using look-ahead approach. Many difficult-resolved branches are due to tandem repeats which are close in the genome. PERGA detects such different copies of the repeats to resolve the branches to make the extension much longer and more accurate. We evaluated PERGA on both Illumina real and simulated datasets ranging from small bacterial genomes to large human chromosome, and it constructed longer and more accurate contigs and scaffolds than other state-of-the-art assemblers. PERGA can be freely downloaded at https://github.com/hitbio/PERGA.     

Optimizing Information in Next-Generation-Sequencing (NGS) Reads for Improving De Novo Genome Assembly

Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.     

The impact of read length on quantification of differentially expressed genes and splice junction detection

BackgroundThe initial next-generation sequencing technologies produced reads of 25 or 36 bp, and only from a single-end of the library sequence. Currently, it is possible to reliably produce 300 bp paired-end sequences for RNA expression analysis. While read lengths have consistently increased, people have assumed that longer reads are more informative and that paired-end reads produce better results than single-end reads. We used paired-end 101 bp reads and trimmed them to simulate different read lengths, and also separated the pairs to produce single-end reads. For each read length and paired status, we evaluated differential expression levels between two standard samples and compared the results to those obtained by qPCR.ResultsWe found that, with the exception of 25 bp reads, there is little difference for the detection of differential expression regardless of the read length. Once single-end reads are at a length of 50 bp, the results do not change substantially for any level up to, and including, 100 bp paired-end. However, splice junction detection significantly improves as the read length increases with 100 bp paired-end showing the best performance. We performed the same analysis on two ENCODE samples and found consistent results confirming that our conclusions have broad application.ConclusionsA researcher could save substantial resources by using 50 bp single-end reads for differential expression analysis instead of using longer reads. However, splicing detection is unquestionably improved by paired-end and longer reads. Therefore, an appropriate read length should be used based on the final goal of the study.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0697-y) contains supplementary material, which is available to authorized users.     

A comprehensive evaluation of assembly scaffolding tools

Background

Genome assembly is typically a two-stage process: contig assembly followed by the use of paired sequencing reads to join contigs into scaffolds. Scaffolds are usually the focus of reported assembly statistics; longer scaffolds greatly facilitate the use of genome sequences in downstream analyses, and it is appealing to present larger numbers as metrics of assembly performance. However, scaffolds are highly prone to errors, especially when generated using short reads, which can directly result in inflated assembly statistics.

Results

Here we provide the first independent evaluation of scaffolding tools for second-generation sequencing data. We find large variations in the quality of results depending on the tool and dataset used. Even extremely simple test cases of perfect input, constructed to elucidate the behaviour of each algorithm, produced some surprising results. We further dissect the performance of the scaffolders using real and simulated sequencing data derived from the genomes of Staphylococcus aureus, Rhodobacter sphaeroides, Plasmodium falciparum and Homo sapiens. The results from simulated data are of high quality, with several of the tools producing perfect output. However, at least 10% of joins remains unidentified when using real data.

Conclusions

The scaffolders vary in their usability, speed and number of correct and missed joins made between contigs. Results from real data highlight opportunities for further improvements of the tools. Overall, SGA, SOPRA and SSPACE generally outperform the other tools on our datasets. However, the quality of the results is highly dependent on the read mapper and genome complexity.     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.     

Paired de bruijn graphs: a novel approach for incorporating mate pair information into genome assemblers

The recent proliferation of next generation sequencing with short reads has enabled many new experimental opportunities but, at the same time, has raised formidable computational challenges in genome assembly. One of the key advances that has led to an improvement in contig lengths has been mate pairs, which facilitate the assembly of repeating regions. Mate pairs have been algorithmically incorporated into most next generation assemblers as various heuristic post-processing steps to correct the assembly graph or to link contigs into scaffolds. Such methods have allowed the identification of longer contigs than would be possible with single reads; however, they can still fail to resolve complex repeats. Thus, improved methods for incorporating mate pairs will have a strong effect on contig length in the future. Here, we introduce the paired de Bruijn graph, a generalization of the de Bruijn graph that incorporates mate pair information into the graph structure itself instead of analyzing mate pairs at a post-processing step. This graph has the potential to be used in place of the de Bruijn graph in any de Bruijn graph based assembler, maintaining all other assembly steps such as error-correction and repeat resolution. Through assembly results on simulated perfect data, we argue that this can effectively improve the contig sizes in assembly.     

Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology

Background

Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging.

Results

We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing.

Conclusions

Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-711) contains supplementary material, which is available to authorized users.     

Scaffolding pre-assembled contigs using SSPACE

SUMMARY: De novo assembly tools play a main role in reconstructing genomes from next-generation sequencing (NGS) data and usually yield a number of contigs. Using paired-read sequencing data it is possible to assess the order, distance and orientation of contigs and combine them into so-called scaffolds. Although the latter process is a crucial step in finishing genomes, scaffolding algorithms are often built-in functions in de novo assembly tools and cannot be independently controlled. We here present a new tool, called SSPACE, which is a stand-alone scaffolder of pre-assembled contigs using paired-read data. Main features are: a short runtime, multiple library input of paired-end and/or mate pair datasets and possible contig extension with unmapped sequence reads. SSPACE shows promising results on both prokaryote and eukaryote genomic testsets where the amount of initial contigs was reduced by at least 75%.     

Dysgu: efficient structural variant calling using short or long reads

Structural variation (SV) plays a fundamental role in genome evolution and can underlie inherited or acquired diseases such as cancer. Long-read sequencing technologies have led to improvements in the characterization of structural variants (SVs), although paired-end sequencing offers better scalability. Here, we present dysgu, which calls SVs or indels using paired-end or long reads. Dysgu detects signals from alignment gaps, discordant and supplementary mappings, and generates consensus contigs, before classifying events using machine learning. Additional SVs are identified by remapping of anomalous sequences. Dysgu outperforms existing state-of-the-art tools using paired-end or long-reads, offering high sensitivity and precision whilst being among the fastest tools to run. We find that combining low coverage paired-end and long-reads is competitive in terms of performance with long-reads at higher coverage values.     

A tool for analyzing mate pairs in assemblies (TAMPA).

The current generation of genome assembly programs uses distance and orientation relationships of paired end reads of clones (mate pairs) to order and orient contigs. Mate pair data can also be used to evaluate and compare assemblies after the fact. Earlier work employed a simple heuristic to detect assembly problems by scanning across an assembly to locate peak concentrations of unsatisfied mate pairs. TAMPA is a novel, computational geometry-based approach to detecting assembly breakpoints by exploiting constraints that mate pairs impose on each other. The method can be used to improve assemblies and determine which of two assemblies is correct in the case of sequence disagreement. Results from several human genome assemblies are presented.     

Generation of Physical Map Contig-Specific Sequences Useful for Whole Genome Sequence Scaffolding

Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.     

Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences

Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/ ).     

Enhanced De Novo Assembly of High Throughput Pyrosequencing Data Using Whole Genome Mapping

Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla _NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.     

A large‐scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large‐scale de novo complementary DNA (cDNA) sequencing effort that acquired ～5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high‐identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large‐scale cDNA dataset for RPW, a much‐needed resource for future molecular studies.     

Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar ‘Fielder’

We have established a high-quality, chromosome-level genome assembly for the hexaploid common wheat cultivar ‘Fielder’, an American, soft, white, pastry-type wheat released in 1974 and known for its amenability to Agrobacterium tumefaciens-mediated transformation and genome editing. Accurate, long-read sequences were obtained using PacBio circular consensus sequencing with the HiFi approach. Sequence reads from 16 SMRT cells assembled using the hifiasm assembler produced assemblies with N50 greater than 20 Mb. We used the Omni-C chromosome conformation capture technique to order contigs into chromosome-level assemblies, resulting in 21 pseudomolecules with a cumulative size of 14.7 and 0.3 Gb of unanchored contigs. Mapping of published short reads from a transgenic wheat plant with an edited seed-dormancy gene, TaQsd1, identified four positions of transgene insertion into wheat chromosomes. Detection of guide RNA sequences in pseudomolecules provided candidates for off-target mutation induction. These results demonstrate the efficiency of chromosome-scale assembly using PacBio HiFi reads and their application in wheat genome-editing studies.