Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods

Bacillus subtilis is the main component in the fermentation of soybeans. To investigate the genetics of the soybean-fermenting B. subtilis strains and its relationship with the productivity of extracellular poly-γ-glutamic acid (γPGA), we sequenced the whole genome of eight B. subtilis stains isolated from non-salted fermented soybean foods in Southeast Asia. Assembled nucleotide sequences were compared with those of a natto (fermented soybean food) starter strain B. subtilis BEST195 and the laboratory standard strain B. subtilis 168 that is incapable of γPGA production. Detected variants were investigated in terms of insertion sequences, biotin synthesis, production of subtilisin NAT, and regulatory genes for γPGA synthesis, which were related to fermentation process. Comparing genome sequences, we found that the strains that produce γPGA have a deletion in a protein that constitutes the flagellar basal body, and this deletion was not found in the non-producing strains. We further identified diversity in variants of the bio operon, which is responsible for the biotin auxotrophism of the natto starter strains. Phylogenetic analysis using multilocus sequencing typing revealed that the B. subtilis strains isolated from the non-salted fermented soybeans were not clustered together, while the natto-fermenting strains were tightly clustered; this analysis also suggested that the strain isolated from “Tua Nao” of Thailand traces a different evolutionary process from other strains.     

Comparative Analysis of Physical Maps of Four Bacillus subtilis (natto) Genomes

The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B. subtilis strain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SPβ, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.     

Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.     

QuorUM: An Error Corrector for Illumina Reads

Motivation

Illumina Sequencing data can provide high coverage of a genome by relatively short (most often 100 bp to 150 bp) reads at a low cost. Even with low (advertised 1%) error rate, 100 × coverage Illumina data on average has an error in some read at every base in the genome. These errors make handling the data more complicated because they result in a large number of low-count erroneous k-mers in the reads. However, there is enough information in the reads to correct most of the sequencing errors, thus making subsequent use of the data (e.g. for mapping or assembly) easier. Here we use the term “error correction” to denote the reduction in errors due to both changes in individual bases and trimming of unusable sequence. We developed an error correction software called QuorUM. QuorUM is mainly aimed at error correcting Illumina reads for subsequent assembly. It is designed around the novel idea of minimizing the number of distinct erroneous k-mers in the output reads and preserving the most true k-mers, and we introduce a composite statistic π that measures how successful we are at achieving this dual goal. We evaluate the performance of QuorUM by correcting actual Illumina reads from genomes for which a reference assembly is available.

Results

We produce trimmed and error-corrected reads that result in assemblies with longer contigs and fewer errors. We compared QuorUM against several published error correctors and found that it is the best performer in most metrics we use. QuorUM is efficiently implemented making use of current multi-core computing architectures and it is suitable for large data sets (1 billion bases checked and corrected per day per core). We also demonstrate that a third-party assembler (SOAPdenovo) benefits significantly from using QuorUM error-corrected reads. QuorUM error corrected reads result in a factor of 1.1 to 4 improvement in N50 contig size compared to using the original reads with SOAPdenovo for the data sets investigated.

Availability

QuorUM is distributed as an independent software package and as a module of the MaSuRCA assembly software. Both are available under the GPL open source license at http://www.genome.umd.edu.

Contact

ude.dmu@siacramg.     

SHRiMP: Accurate Mapping of Short Color-space Reads

The development of Next Generation Sequencing technologies, capable of sequencing hundreds of millions of short reads (25–70 bp each) in a single run, is opening the door to population genomic studies of non-model species. In this paper we present SHRiMP - the SHort Read Mapping Package: a set of algorithms and methods to map short reads to a genome, even in the presence of a large amount of polymorphism. Our method is based upon a fast read mapping technique, separate thorough alignment methods for regular letter-space as well as AB SOLiD (color-space) reads, and a statistical model for false positive hits. We use SHRiMP to map reads from a newly sequenced Ciona savignyi individual to the reference genome. We demonstrate that SHRiMP can accurately map reads to this highly polymorphic genome, while confirming high heterozygosity of C. savignyi in this second individual. SHRiMP is freely available at http://compbio.cs.toronto.edu/shrimp.     

FastUniq: A Fast De Novo Duplicates Removal Tool for Paired Short Reads

The presence of duplicates introduced by PCR amplification is a major issue in paired short reads from next-generation sequencing platforms. These duplicates might have a serious impact on research applications, such as scaffolding in whole-genome sequencing and discovering large-scale genome variations, and are usually removed. We present FastUniq as a fast de novo tool for removal of duplicates in paired short reads. FastUniq identifies duplicates by comparing sequences between read pairs and does not require complete genome sequences as prerequisites. FastUniq is capable of simultaneously handling reads with different lengths and results in highly efficient running time, which increases linearly at an average speed of 87 million reads per 10 minutes. FastUniq is freely available at http://sourceforge.net/projects/fastuniq/.     

Comparative Analysis of Functional Metagenomic Annotation and the Mappability of Short Reads

To assess the functional capacities of microbial communities, including those inhabiting the human body, shotgun metagenomic reads are often aligned to a database of known genes. Such homology-based annotation practices critically rely on the assumption that short reads can map to orthologous genes of similar function. This assumption, however, and the various factors that impact short read annotation, have not been systematically evaluated. To address this challenge, we generated an extremely large database of simulated reads (totaling 15.9 Gb), spanning over 500,000 microbial genes and 170 curated genomes and including, for many genomes, every possible read of a given length. We annotated each read using common metagenomic protocols, fully characterizing the effect of read length, sequencing error, phylogeny, database coverage, and mapping parameters. We additionally rigorously quantified gene-, genome-, and protocol-specific annotation biases. Overall, our findings provide a first comprehensive evaluation of the capabilities and limitations of functional metagenomic annotation, providing crucial goal-specific best-practice guidelines to inform future metagenomic research.     

Improving PacBio Long Read Accuracy by Short Read Alignment

The recent development of third generation sequencing (TGS) generates much longer reads than second generation sequencing (SGS) and thus provides a chance to solve problems that are difficult to study through SGS alone. However, higher raw read error rates are an intrinsic drawback in most TGS technologies. Here we present a computational method, LSC, to perform error correction of TGS long reads (LR) by SGS short reads (SR). Aiming to reduce the error rate in homopolymer runs in the main TGS platform, the PacBio® RS, LSC applies a homopolymer compression (HC) transformation strategy to increase the sensitivity of SR-LR alignment without scarifying alignment accuracy. We applied LSC to 100,000 PacBio long reads from human brain cerebellum RNA-seq data and 64 million single-end 75 bp reads from human brain RNA-seq data. The results show LSC can correct PacBio long reads to reduce the error rate by more than 3 folds. The improved accuracy greatly benefits many downstream analyses, such as directional gene isoform detection in RNA-seq study. Compared with another hybrid correction tool, LSC can achieve over double the sensitivity and similar specificity.     

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome

Background

Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base.

Results

We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly.

Conclusions

SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0726-6) contains supplementary material, which is available to authorized users.     

Haplotype-Phased Synthetic Long Reads from Short-Read Sequencing

Next-generation DNA sequencing has revolutionized the study of biology. However, the short read lengths of the dominant instruments complicate assembly of complex genomes and haplotype phasing of mixtures of similar sequences. Here we demonstrate a method to reconstruct the sequences of individual nucleic acid molecules up to 11.6 kilobases in length from short (150-bp) reads. We show that our method can construct 99.97%-accurate synthetic reads from bacterial, plant, and animal genomic samples, full-length mRNA sequences from human cancer cell lines, and individual HIV env gene variants from a mixture. The preparation of multiple samples can be multiplexed into a single tube, further reducing effort and cost relative to competing approaches. Our approach generates sequencing libraries in three days from less than one microgram of DNA in a single-tube format without custom equipment or specialized expertise.     

Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling

Background

Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecific comparisons. Currently, different platforms for massively parallel sequencing are available, which differ in read length and sequencing costs. The 454-technology offers the highest read length. The other sequencing technologies are more cost effective, on the expense of shorter reads. Reliable expression profiling by massively parallel sequencing depends crucially on the accuracy to which the reads could be mapped to the corresponding genes.

Methodology/Principal Findings

We performed an in silico analysis to evaluate whether incorrect mapping of the sequence reads results in a biased expression pattern. A comparison of six available mapping software tools indicated a considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we found that for compact genomes both short (35 bp, 50 bp) and long sequence reads (100 bp) result in an almost unbiased expression pattern. In contrast, for species with a larger genome containing more gene families and repetitive DNA, shorter reads (35–50 bp) produced a considerable bias in gene expression. In humans, about 10% of the genes had fewer than 50% of the sequence reads correctly mapped. Sequence polymorphism up to 9% had almost no effect on the mapping accuracy of 100 bp reads. For 35 bp reads up to 3% sequence divergence did not affect the mapping accuracy strongly. The effect of indels on the mapping efficiency strongly depends on the mapping software.

Conclusions/Significance

In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable bias due to incorrectly mapped sequence reads if the read length is short. Nevertheless, this bias could be accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect the mapping accuracy and may cause a biased gene expression measurement. The choice of the mapping software is highly critical and the reliability depends on the presence/absence of indels and the divergence between reads and the reference genome. Overall, we found SSAHA2 and CLC to produce the most reliable mapping results.     

Genome assembly using Nanopore-guided long and error-free DNA reads

Background

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.

Results

The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.

Conclusions

We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1519-z) contains supplementary material, which is available to authorized users.     

High-quality Arabidopsis thaliana Genome Assembly with Nanopore and HiFi Long Reads

Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains a significant number of missing segments. Here, we reported a high-quality and almost complete Col-0 genome assembly with two gaps (named Col-XJTU) by combining the Oxford Nanopore Technologies ultra-long reads, Pacific Biosciences high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of the Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of the TAIR10.1 reference (ranging from 45 to 52). We completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 was completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled a 3.8-Mb-long CEN1 and a 3.5-Mb-long CEN2. We also investigated the structure and epigenetics of centromeres. Four clusters of CEN180 monomers were detected, and the centromere-specific histone H3-like protein (CENH3) exhibited a strong preference for CEN180 Cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as the genetic and epigenetic features in plants.     

IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

Motivation

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads.

Results

We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity.

Availability and Implementation

IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.     

Establishment of an eHAP1 human haploid cell line hybrid reference genome assembled from short and long reads

Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line.     

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N₅₀ of 10,384 bp and scaffold N₅₀ of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.     

Construction of a Kit of Reference Strains for Rapid Genetic Mapping in Bacillus subtilis 168

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.